Four-helical bundle protein zsig81

ABSTRACT

This present invention is directed to polypeptide and polynucleotide molecules that encode a four-helical bundle cytokine. The cytokine has been designated zsig81, and has restricted expression in primarily heart, lung and liver. zsig81 has been shown to stimulate proliferation of hematopoietic cells and will be useful expansion of these cells, as well as conditions associated with hematopoietic cells. The invention is directed to antibodies and methods of making zsig81 polypeptides, as well.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application 09/585,228which is related to Provisional Application 60/137,057, filed on Jun. 1,1999. Under 35 U.S.C. § 119(e)(1), this application claims benefit ofsaid Provisional Application.

BACKGROUND OF THE INVENTION

[0002] Cellular differentiation of multicellular organisms is controlledby hormones and polypeptide growth factors. These diffusable moleculesallow cells to communicate with each other and act in concert to formtissues and organs, and to repair and regenerate damaged tissue.Examples of hormones and growth factors include the steroid hormones,parathyroid hormone, follicle stimulating hormone, the interferons, theinterleukins, platelet derived growth factor, epidermal growth factor,and granulocyte-macrophage colony stimulating factor, among others.

[0003] Hormones and growth factors influence cellular metabolism bybinding to receptor proteins. Certain receptors are integral membraneproteins that bind with the hormone or growth factor outside the cell,and that are linked to signaling pathways within the cell, such assecond messenger systems. Other classes of receptors are solubleintracellular molecules.

[0004] In general, there is conservation among the cytokine receptors,which are classified into two classes. The cytokine ligands for thesereceptors have some conserved structural identity, as well, however, thebiological activity of these ligands is diverse. The molecules of thepresent invention belong to a structural class of ligands that ischaracterized by comprising a four-helical bundle. As a group, thecytokine family of ligands have been extremely valuable as therapeuticsand reagents for understanding the growth and maturation of many celltypes. The cells influenced by cytokines range from totipotent stemcells to terminally differentiated cells with specialized functionscritical to homeostasis of a broad spectrum of living systems. Thus,based on the activities of the cytokine family of proteins there is aneed for new cytokines, cytokine agonists and cytokine antagonists, aswell as related compounds and methods. The present invention providessuch polypeptides for these and other uses that should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The FIGURE illustrates a Hopps Woods Hydrophobicity Plot.

DETAILED DESCRIPTION OF THE INVENTION

[0006] Prior to setting forth the invention in detail, it may be helpfulto the understanding thereof to define the following terms:

[0007] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0008] The term “allelic variant” is used herein to denote any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

[0009] The terms “amino-terminal” and “carboxyl-terminal” are usedherein to denote positions within polypeptides. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide to denote proximity or relative position. Forexample, a certain sequence positioned carboxyl-terminal to a referencesequence within a polypeptide is located proximal to the carboxylterminus of the reference sequence, but is not necessarily at thecarboxyl terminus of the complete polypeptide.

[0010] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0011] The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence. For example, thesequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0012] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0013] The term “expression vector” is used to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

[0014] The term “isolated”, when applied to a polynucleotide, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985).

[0015] An “isolated” polypeptide or protein is a polypeptide or proteinthat is found in a condition other than its native environment, such asapart from blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

[0016] The term “operably linked”, when referring to DNA segments,indicates that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiates inthe promoter and proceeds through the coding segment to the terminator.

[0017] The term “ortholog” denotes a polypeptide or protein obtainedfrom one species that is the functional counterpart of a polypeptide orprotein from a different species. Sequence differences among orthologsare the result of speciation.

[0018] “Paralogs” are distinct but structurally related proteins made byan organism. Paralogs are believed to arise through gene duplication.For example, α-globin, β-globin, and myoglobin are paralogs of eachother.

[0019] A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

[0020] A “polypeptide” is a polymer of amino acid residues joined bypeptide bonds, whether produced naturally or synthetically. Polypeptidesof less than about 10 amino acid residues are commonly referred to as“peptides”.

[0021] The term “promoter” is used herein for its art-recognized meaningto denote a portion of a gene containing DNA sequences that provide forthe binding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

[0022] A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

[0023] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

[0024] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger polypeptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0025] The term “splice variant” is used herein to denote alternativeforms of RNA transcribed from a gene. Splice variation arises naturallythrough use of alternative splicing sites within a transcribed RNAmolecule, or less commonly between separately transcribed RNA molecules,and may result in several mRNAs transcribed from the same gene. Splicevariants may encode polypeptides having altered amino acid sequence. Theterm splice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

[0026] Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

[0027] All references cited herein are incorporated by reference intheir entirety.

[0028] The present invention is based in part upon the discovery of anovel DNA sequence that encodes a four-helical bundle cytokine. Thefamily of four-helical bundle cytokines is described herein and thepolynucleotides and polypeptides of the present invention will comprisesome or all of the components characterizing the protein family. Thepolypeptides of the present invention have been designated zsig81.

[0029] Sequence analysis revealed an open reading frame of 173 aminoacids, with a secretory signal peptide of 17 amino acids at theN-terminus and a mature protein of 156 amino acids. Based on N-terminalsequence analysis of recombinantly expressed human protein, cleavageafter residue 4 (His) results in a biologically active molecule of 151amino acids as shown in SEQ ID NO: 2 from residue 5 (Arg) to residue 156(Pro). Four-helical cytokine polypeptides generally have a strong signalsequence with a proximal upstream stop codon. In the molecules of thepresent invention, the human zsig81 encodes a stop codon beginning atnucleotide −30 as shown in SEQ ID NO: 1. The corresponding nucleotide inthe mouse ortholog of zsig81 can be found at nucleotide −27 of SEQ IDNO: 3. Those skilled in the art will recognize, however, that somecytokines (e.g., endothelial cell growth factor, basic FGF, and IL-1β)do not comprise conventional secretory peptides and are secreted by amechanism that is not understood. The cDNA also includes a clearpolyadenylation signal, as well as two message instability motifs(ATTTA) in the 3′-untranslated region. These message instability motifsare characteristic of cytokine genes (Shaw and Kamen, Cell 46:659-667,1986).

[0030] In general, cytokines are predicted to have a four-alpha helixstructure, with helices A, C and D being most important inligand-receptor interactions, containing conserved motifs among membersof the family. The four helices, designated A, B, C, and D have beenpredicted to span amino acid residues 30 to 44 (helix A), 56-70 (helixB), 86-94 (helix C) and 135-149 (helix D), as shown in SEQ ID NO: 2.Helix C boundaries are predicted to either span residues 80-94 or 86-100of SEQ ID NO: 2. A structural analysis indicates that the loop structureof zsig81 conforms with other members of the cytokine family in that theA/B loop is long, the B/C loop is short, and the C/D loop is long. Thisloop structure results in an up-up-down-down helical organization,typical of cytokines. Therefore, the predicted helical structure ofzsig81 would include the molecule in the family of short-helix formcytokines with IL-2, IL-4, IL-5, and GM-CSF. Studies using CNTF and IL-6demonstrated that a CNTF helix can be exchanged for the equivalent helixin IL-6, conferring CTNF-binding properties to the chimera. Thus, itappears that functional domains of four-helical cytokines are determinedon the basis of structural homology, irrespective of sequence identity,and can maintain functional integrity in a chimera (Kallen et al., J.Biol. Chem. 274:11859-11867, 1999). Therefore, the helical domains ofzsig81 will be useful for preparing chimeric fusion molecules,particularly with other short-helix form cytokines to determine andmodulate receptor binding specificity. Of particular interest are fusionproteins engineered with helix A and/or helix D, and fusion proteinsthat combine helical and loop domains from other short-form cytokinessuch as IL-2, IL-4, IL-15 and GM-CSF. In the amino acid sequenceN-terminal to helix A and C-terminal to the secretory signal sequence,there is a proline-rich region. C-terminal to helix D, the amino acidsequence forms a short tail spanning residues 150 to 156. Within thehelical regions, residues that are expected to lie within the core ofthe four-helix bundle occur at residues 30, 33, 34, 37, 40, 41, 44, 56,59, 60, 63, 66, 67,70, 80, 83, 84, 87, 90, 91, 94, 135, 138, 139, 142,145, 146, and 149.

[0031] Zsig81 has two Cysteine residues, located at residue 3, which isN-terminal to loop A/B, and at residue 76, which is located in the B/Cloop. However, when protein that has been expressed in baculovirus isN-terminally sequenced, the biologically active molecule is aheterogeneous mixture with a significant portion of the protein cleavedafter His4, thereby resulting in a molecule with a single cysteine atresidue 76. In the mature zsig81 molecule, the two cysteines may form anintramolecular disulfide bond; and in the N-terminally truncatedmolecule or mature molecule, a single cysteine may form homodimers orheterodimers with another binding partner. Using SDS-PAGE analysis, ahomodimeric form of zsig81 protein has been detected. Based on analysisof amino acid charges, the helix A is negatively charged and the C helixis positively charged. The predicted molecular weight of zsig81 is17,533 Daltons, and there is no apparent glycosylation.

[0032] Analysis of the tissue distribution of the human mRNAcorresponding to this novel DNA showed that expression was found to berestricted to heart and liver. Within the heart tissue, the highestexpression of human MRNA has been localized to the smooth muscle ofaorta. While the cDNA is predicted to be approximately 1.7 kb in size,bands identified on Northerns were approximately 5.0 kb. A splicevariant containing approximately 2.5 kb 3′ untranslated region wasidentified in human zsig81 mRNA, and is believed to correspond to the5.0 kb band seen by Northern analysis.

[0033] The ortholog from mouse has also been identified and designatedzsig81m. A DNA sequence and corresponding putative amino acid sequenceare shown in SEQ ID NOS: 3 and 4, respectively.

[0034] SEQ ID NO: 5 is a degenerate polynucleotide sequence thatencompasses all polynucleotides that could encode the zsig81 polypeptideof SEQ ID NO: 2 (amino acids 1 or 24 to 354). Thus, zsig81polypeptide-encoding polynucleotides ranging from nucleotide 134 or 185to nucleotide 655 of SEQ ID NO: 2 or nucleotide 1 or 52 to 519 of SEQ IDNO: 5 are contemplated by the present invention. Also contemplated bythe present invention are fragments and fusions as described herein withrespect to SEQ ID NO: 1, which are formed from analogous regions of SEQID NO: 5, wherein nucleotides 134 to 184 of SEQ ID NO: 1 correspond tonucleotides 1 to 51 of SEQ ID NO: 5, for the secretory signal sequence;wherein nucleotides 272 to 316 of SEQ ID NO: 1 correspond to nucleotides139 to 183 of SEQ ID NO: 5, for helix A; wherein nucleotides 350 to 394of SEQ ID NO: 1 correspond to nucleotides 216 to 261 of SEQ ID NO: 5 forhelix B; wherein nucleotides 440 to 466 of SEQ ID NO: 1 correspond tonucleotides 307 to 331 of SEQ ID NO: 5, for helix C; and whereinnucleotide 587 to nucleotide 631 of SEQ ID NO: 1 correspond tonucleotide 454 to nucleotide 499 of SEQ ID NO: 5 for helix D. Table 1sets forth the one-letter codes used within SEQ ID NO: 5 to denotedegenerate nucleotide positions. “Resolutions” are the nucleotidesdenoted by a code letter. “Complement” indicates the code for thecomplementary nucleotide(s). For example, the code Y denotes either C orT, and its complement R denotes A or G, A being complementary to T, andG being complementary to C. TABLE 1 Nucleotide Resolution ComplementResolution A A T T C C G G G G C C T I A A R A|G Y C|T Y C|T R A|G M A|CK G|T K G|T M A|C S G|G S G|G W A|T W A|T H A|C|T D A|G|T B C|G|T VA|C|G V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T

[0035] The degenerate codons used in SEQ ID NOS: 2 and 4, encompassingall 5 possible codons for a given amino acid, are set forth in Table 2.

[0036] One of ordinary skill in the art will appreciate that someambiguity is introduced in determining a degenerate codon,representative of all possible codons encoding each amino acid. Forexample, the degenerate codon for serine (WSN) can, in somecircumstances, encode arginine (AGR), and the degenerate codon forarginine (MGN) can, in some circumstances, encode serine (AGY). Asimilar relationship exists between codons encoding phenylalanine andleucine. Thus, some polynucleotides encompassed by the degeneratesequence may encode variant amino acid sequences, but one of ordinaryskill in the art can easily identify such variant sequences by referenceto the amino acid sequence of SEQ ID NOS: 2 and 4. Variant sequences canbe readily tested for functionality as described herein.

[0037] One of ordinary skill in the art will also appreciate thatdifferent species can exhibit “preferential codon usage.” In general,see, Grantham, et al., Nuc. Acids Res., 8:1893-912, 1980; Haas, et al.Curr. Biol., 6:315-24, 1996; Wain-Hobson, et al., Gene, 13:355-64, 1981;Grosjean and Fiers, Gene, 18:199-209, 1982; Holm, Nuc. Acids Res.,14:3075-87, 1986; Ikemura, J. Mol. Biol., 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO: 5 serves as a template for optimizing expression of polynucleotidesin various cell types and species commonly used in the art and disclosedherein. Sequences containing preferential codons can be tested andoptimized for expression in various species, and tested forfunctionality as disclosed herein.

[0038] The present invention further provides variant polypeptides andnucleic acid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are zsig81 polypeptidesfrom other mammalian species, including murine, porcine, ovine, bovine,canine, feline, equine, and other primate polypeptides. Orthologs ofhuman zsig81 can be cloned using information and compositions providedby the present invention in combination with conventional cloningtechniques. For example, a cDNA can be cloned using mRNA obtained from atissue or cell type that expresses zsig81 as disclosed herein. Suitablesources of mRNA can be identified by probing northern blots with probesdesigned from the sequences disclosed herein. A library is then preparedfrom mRNA of a positive tissue or cell line. The mouse sequence zsig81is a representative ortholog of the human zsig81, and is disclosedherein as SEQ ID NOS:3 and 4.

[0039] An zsig81-encoding cDNA can then be isolated by a variety ofmethods, such as by probing with a complete or partial human cDNA orwith one or more sets of degenerate probes based on the disclosedsequences. A cDNA can also be cloned using the polymerase chain reactionwith primers designed from the representative human zsig81 sequencesdisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to zsig81 polypeptide. Similartechniques can also be applied to the isolation of genomic clones.

[0040] The present invention provides polynucleotide molecules includingDNA and RNA molecules that encode the zsig81 polypeptides disclosedabove.

[0041] Zsig81 polynucleotide sequences disclosed herein can also be usedas probes or primers to clone 5′ non-coding regions of a zsig81 gene. Inview of the tissue-specific expression observed for zsig81 by Northernblotting, this gene region is expected to provide for heart- andliver-specific expression. Promoter elements from a zsig81 gene couldthus be used to direct the tissue-specific expression of heterologousgenes in, for example, transgenic animals or patients treated with genetherapy. Cloning of 5′ flanking sequences also facilitates production ofzsig81 proteins by “gene activation” as disclosed in U.S. Pat. No.5,641,670. Briefly, expression of an endogenous zsig81 gene in a cell isaltered by introducing into the zsig81 locus a DNA construct comprisingat least a targeting sequence, a regulatory sequence, an exon, and anunpaired splice donor site. The targeting sequence is a zsig81 5′non-coding sequence that permits homologous recombination of theconstruct with the endogenous zsig81 locus, whereby the sequences withinthe construct become operably linked with the endogenous zsig81 codingsequence. In this way, an endogenous zsig81 promoter can be replaced orsupplemented with other regulatory sequences to provide enhanced,tissue-specific, or otherwise regulated expression.

[0042] Those skilled in the art will recognize that the sequencedisclosed in SEQ ID NO:1 represents a single allele of human zsig81 andthat allelic variation and alternative splicing are expected to occur.Allelic variants of this sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures. Allelic variants of the nucleotide sequence shown in SEQ IDNO:1, including those containing silent mutations and those in whichmutations result in amino acid sequence changes, are within the scope ofthe present invention, as are proteins which are allelic variants of SEQID NO:2. cDNA molecules generated from alternatively spliced mRNAs,which retain the properties of the zsig81 polypeptide are includedwithin the scope of the present invention, as are polypeptides encodedby such cDNAs and mRNAs. Allelic variants and splice variants of thesesequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals or tissues 10 according to standard proceduresknown in the art.

[0043] The precise knowledge of a gene's position can be useful for anumber of purposes, including: 1) determining if a sequence is part ofan existing contig and obtaining additional surrounding geneticsequences in various forms, such as YACs, BACs or cDNA clones; 2)providing a possible candidate gene for an inheritable disease whichshows linkage to the same chromosomal region; and 3) cross-referencingmodel organisms, such as mouse, which may aid in determining whatfunction a particular gene might have.

[0044] Analysis of chromosomal DNA using the zsig81 polynucleotidesequence is useful for correlating disease with abnormalities localizedto chromosome 7. The human zsig81 gene has been localized to chromosome7q32-q33. Use as a diagnostic could assist physicians in determining thetype of disease and appropriate associated therapy, or could assist ingenetic counseling. As such, the inventive anti-zsig81 antibodies,polynucleotides, and polypeptides can be used for the detection ofzsig81 polypeptide, mRNA or anti-zsig81 antibodies, thus serving asmarkers and be directly used for detecting genetic diseases or cancers,as described herein, using methods known in the art and describedherein. Further, zsig81 polynucleotide probes can be used to detectabnormalities involving chromosome 7q32-q33 as described herein. Theseabnormalities may be associated with human diseases, or tumorigenesis,spontaneous abortion or other genetic disorders. Thus, zsig81polynucleotide probes can be used to detect abnormalities or genotypesassociated with these defects.

[0045] As discussed above, defects in the zsig81 gene itself may resultin a heritable human disease state. Molecules of the present invention,such as the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment of diseases associated with a zsig81genetic defect. In addition, zsig81 polynucleotide probes can be used todetect allelic differences between diseased or non-diseased individualsat the zsig81 chromosomal locus. As such, the zsig81 sequences can beused as diagnostics in forensic DNA profiling.

[0046] In general, the diagnostic methods used in genetic linkageanalysis, to detect a genetic abnormality or aberration in a patient,are known in the art. Most diagnostic methods comprise the steps of (i)obtaining a genetic sample from a potentially diseased patient, diseasedpatient or potential non-diseased carrier of a recessive disease allele;(ii) producing a first reaction product by incubating the genetic samplewith a zsig81 polynucleotide probe wherein the polynucleotide willhybridize to complementary polynucleotide sequence, such as in RFLPanalysis or by incubating the genetic sample with sense and antisenseprimers in a PCR reaction under appropriate PCR reaction conditions;(iii) Visualizing the first reaction product by gel electrophoresisand/or other known method such as visualizing the first reaction productwith a zsig81 polynucleotide probe wherein the polynucleotide willhybridize to the complementary polynucleotide sequence of the firstreaction; and (iv) comparing the visualized first reaction product to asecond control reaction product of a genetic sample from a normal orcontrol individual. A difference between the first reaction product andthe control reaction product is indicative of a genetic abnormality inthe diseased or potentially diseased patient, or the presence of aheterozygous recessive carrier phenotype for a non-diseased patient, orthe presence of a genetic defect in a tumor from a diseased patient, orthe presence of a genetic abnormality in a fetus or pre-implantationembryo. For example, a difference in restriction fragment pattern,length of PCR products, length of repetitive sequences at the zsig81genetic locus, and the like, are indicative of a genetic abnormality,genetic aberration, or allelic difference in comparison to the normalcontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated from any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO:1, thecomplement of SEQ ID NO:1, or an RNA equivalent thereof. Such methods ofshowing genetic linkage analysis to human disease phenotypes are wellknown in the art. For reference to PCR based methods in diagnostics see,generally, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), White (ed.), PCR Protocols: Current Methods andApplications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

[0047] Mutations associated with the zsig81 locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc. 1998).Direct analysis of an zsig81 gene for a mutation can be performed usinga subject's genomic DNA. Methods for amplifying genomic DNA, obtainedfor example from peripheral blood lymphocytes, are well-known to thoseof skill in the art (see, for example, Dracopoli et al. (eds.), CurrentProtocols in Human Genetics, at pages 7.1.6 to 7.1.7 (John Wiley & Sons1998)).

[0048] Positions of introns in the mouse zsig81 gene were determined byidentification of genomic clones, followed by sequencing the intron/exonjunctions. The coding regions for the zsig81 molecule are contained inthree exons. The first intron junction lies between amino acid residue 8(Arg) and residue 9 (Ala) in Seq. ID. No. 4.

[0049] The second intron junction lies between amino acid residue 42(Glu) and residue 43 (Leu) in Seq. ID. No. 4. The third intron containsthe remaining DNA encoding for the C-terminal portion of the molecule,as well as a 3′ untranslated region.

[0050] Within an embodiment of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules having the nucleotide sequence of SEQ ID NO: 1 from nucleotide134 or 185 to 655, or the full length sequence, to nucleic acidmolecules having a nucleotide sequence complementary to SEQ ID NO: 1. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe.

[0051] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA andDNA-RNA, can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

[0052] It is well within the abilities of one skilled in the art toadapt these conditions for use with a particular polynucleotide hybrid.The T_(m) for a specific target sequence is the temperature (underdefined conditions) at which 50% of the target sequence will hybridizeto a perfectly matched probe sequence. Those conditions which influencethe T_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

[0053] Following hybridization, the nucleic acid molecules can be washedto remove non-hybridized nucleic acid molecules under stringentconditions, or under highly stringent conditions. Typical stringentwashing conditions include washing in a solution of 0.5×-2×SSC with 0.1%sodium dodecyl sulfate (SDS) at 55-65° C. That is, nucleic acidmolecules encoding a variant zsig81 polypeptide hybridize with a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.,including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65°C. One of skill in the art can readily devise equivalent conditions, forexample, by substituting SSPE for SSC in the wash solution.

[0054] Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variantzsig81 polypeptide hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1%SDS at 50° C., or 0.2×SSC with 0.1% SDS at 65° C.

[0055] The present invention also provides isolated zsig81 polypeptidesthat have a substantially similar sequence identity to the polypeptidesof SEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides comprising atleast 70% to 80%, and in certain embodiments at least 90% to 95%, or inother embodiments greater than 95% sequence identity to the sequencesshown in SEQ ID NO:2, or their orthologs. The present invention alsoincludes polypeptides that comprise an amino acid sequence having atleast 70% to 80%, and in certain embodiments at least 90% to 95%, or inother embodiments greater than 95% sequence identity to the sequence ofamino acid residues 1 or 24 to 354 of SEQ ID NO:2. The present inventionfurther includes nucleic acid molecules that encode such polypeptides.Methods for determining percent identity are described below.

[0056] The present invention also contemplates zsig81 variant nucleicacid molecules that can be identified using two criteria: adetermination of the similarity between the encoded polypeptide with theamino acid sequence of SEQ ID NO:2, and/or a hybridization assay, asdescribed above. Such zsig81 variants include nucleic acid molecules (1)that hybridize with a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1 (or its complement) under stringent washingconditions, in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C., or (2) that encode a polypeptide having atleast 70%, at least 80%, at least 90%, at least 95% or greater than 95%sequence identity to the amino acid sequence of SEQ IDNO:2.Alternatively, zsig81 variants can be characterized as nucleic acidmolecules (1) that hybridize with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) that encode apolypeptide having at least 70% to 80%, and in certain embodiments atleast 90% to 95%, or in other embodiments greater than 95% sequenceidentity to the amino acid sequence of SEQ ID NO:2.

[0057] Percent sequence identity is determined by conventional methods.See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes).$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}\quad {number}\quad {of}\quad {gaps}\quad {introduced}} \right. \\\left. {{into}\quad {the}\quad {longer}\quad {sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0058] Those skilled in the art appreciate that there are manyestablished algorithms available to align two amino acid sequences. The“FASTA” similarity search algorithm of Pearson and Lipman is a suitableprotein alignment method for examining the level of identity shared byan amino acid sequence disclosed herein and the amino acid sequence of aputative variant zsig81. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

[0059] Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2) anda test sequence that have either the highest density of identities (ifthe ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

[0060] FASTA can also be used to determine the sequence identity ofnucleic acid molecules using a ratio as disclosed above. For nucleotidesequence comparisons, the ktup value can range between one to six,preferably from four to six.

[0061] Variant zsig81 polypeptides or polypeptides with substantiallysimilar sequence identity are characterized as having one or more aminoacid substitutions, deletions or additions. These changes are preferablyof a minor nature, that is conservative amino acid substitutions (seeTable 4) and other substitutions that do not significantly affect thefolding or activity of the polypeptide; small deletions, typically ofone to about 30 amino acids; and amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides of from about 28 to 354 amino acid residuesthat comprise a sequence that is at least 70% to 80%, and in certainembodiments at least 90% to 95%, or in other embodiments greater than95% or more identical to the corresponding region of SEQ ID NO:2. Inparticular, peptides and polypeptides corresponding to regions of thezsig81 molecules as shown in SEQ ID NO: 2 include the helix A (residues30-44), helix B (residues 56-70), helix C (comprising residues 86-94)and helix D (residues 135-149) are within the scope of the presentinvention. Polypeptides comprising affinity tags can further comprise aproteolytic cleavage site between the zsig81 polypeptide and theaffinity tag. Preferred such sites include thrombin cleavage sites andfactor Xa cleavage sites. TABLE 4 Conservative amino acid substitutionsBasic: arginine lysine histidine Acidic: glutamic acid aspartic acidPolar: glutamine asparagine Hydrophobic: leucine isoleucine valineAromatic: phenylalanine tryptophan tyrosine Small: glycine alanineserine threonine methionine

[0062] The proteins of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and arminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

[0063] In a second method, translation is carried out in Xenopus oocytesby microinjection of mutated mRNA and chemically aminoacylatedsuppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)).Within a third method, E. coli cells are cultured in the absence of anatural amino acid that is to be replaced (e.g., phenylalanine) and inthe presence of the desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

[0064] A limited number of non-conservative amino acids, amino acidsthat are not encoded by the genetic code, non-naturally occurring aminoacids, and unnatural amino acids may be substituted for zsig81 aminoacid residues.

[0065] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

[0066] Variants of the disclosed zsig81 nucleotide and polypeptidesequences can also be generated through DNA shuffling as disclosed byStemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA91:10747 (1994), and international publication No. WO 97/20078. Briefly,variant DNA molecules are generated by in vitro homologous recombinationby random fragmentation of a parent DNA followed by reassembly usingPCR, resulting in randomly introduced point mutations. This techniquecan be modified by using a family of parent DNA molecules, such asallelic variants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

[0067] Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, peptides thatbind to and inhibit activation of zsig81 receptor, or polypeptides thatbind with anti-zsig81 antibodies, can be recovered from the host cellsand rapidly sequenced using modern equipment. These methods allow therapid determination of the importance of individual amino acid residuesin a polypeptide of interest, and can be applied to polypeptides ofunknown structure.

[0068] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'lAcad. Sci. USA 88:4498 (1991), Coombs and Corey, “Site-DirectedMutagenesis and Protein Engineering,” in Proteins: Analysis and Design,Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)). In thelatter technique, single alanine mutations are introduced at everyresidue in the molecule, and the resultant mutant molecules are testedfor biological activity as disclosed below to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699 (1996). The identities ofessential amino acids can also be inferred from analysis of homologieswith zsig81.

[0069] The location of zsig81 receptor binding domains can be identifiedby physical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306(1992), Smith et al., J. Mol. Biol. 224:899 (1992), and Wlodaver et al.,FEBS Lett. 309:59 (1992). Moreover, zsig81 labeled with biotin or FITCcan be used for expression cloning of zsig81 receptors.

[0070] The present invention also includes “functional fragments” ofzsig81 polypeptides and nucleic acid molecules encoding such functionalfragments. As previously described herein, zsig81 is characterized by afour-helical bundle. Thus, the present invention further provides fusionproteins encompassing (a) polypeptide molecules comprising one or moreof the regions described above, and (b) biologically active fragmentscomprising portions of one or more of the domains. The other polypeptidemay be another regions from another cytokine, a non-native and/or anunrelated secretory signal peptide to facilitate secretion of the fusionprotein. Thus, as described herein functional domains of zsig81 will beuseful for preparing chimeric fusion proteins that combine helical loopdomains from other short form cytokines. Such chimeras will havespecificity determined by the component regions used (Kallen et al,ibid., 1999). Chimeric molecules can be prepared using one or morehelices of secretory signal sequence from zsig81 in combination withcorresponding regions from other cytokines.

[0071] Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes an zsig81 polypeptide. As an illustration, DNA molecules havingthe nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for zsig81, or for theability to bind anti-zsig81 antibodies. One alternative to exonucleasedigestion is to use oligonucleotide-directed mutagenesis to introducedeletions or stop codons to specify production of a desired fragment.Alternatively, particular fragments of an zsig81 gene can be synthesizedusing the polymerase chain reaction.

[0072] Standard methods for identifying functional domains arewell-known to those of skill in the art. For example, studies on thetruncation at either or both termini of interferons have been summarizedby Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-SA synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987), Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation, Vol. 1, Boynton etal., (eds.) pages 169-199 (Academic Press 1985), Coumailleau et al., J.Biol. Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995);

[0073] Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995), and Meiselet al., Plant Molec. Biol. 30:1 (1996).

[0074] The present invention also contemplates functional fragments ofan zsig81 gene that has amino acid changes, compared with the amino acidsequence of SEQ ID NO:2. A variant zsig81 gene can be identified on thebasis of structure by determining the level of identity with nucleotideand amino acid sequences of SEQ ID NOs:1 and 2, as discussed above. Analternative approach to identifying a variant gene on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant zsig81 gene can hybridize to a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, as discussed above.

[0075] Amino acid sequence changes are made in zsig81 polypeptides so asto minimize disruption of higher order structure essential to biologicalactivity. For example, when the zsig81 polypeptide comprises one or morehelices, changes in amino acid residues will be made so as not todisrupt the loop length and conformation characteristics of the proteinfamily. The effects of amino acid sequence changes can be predicted by,for example, computer modeling as disclosed above or determined byanalysis of crystal structure (see, e.g., Lapthorn et al., ibid.). Othertechniques that can be used, independently or in combination, to analyzeand compare the structural features that affect folding of a variantprotein or polypeptide to a standard molecule to determine whether suchmodifications would be significant are well known in the art. Forexample, comparison of the cysteine pattern in a variant and standardmolecule can be made. Mass spectrometry and chemical modification usingreduction and alkylation provide methods for determining cysteineresidues which are associated with disulfide bonds or are free of suchassociations (Bean et al., Anal. Biochem. 201:216-226, 1992; Gray,Protein Sci. 2:1732-1748, 1993; and Patterson et al., Anal. Chem.66:3727-3732, 1994). It is generally believed that if a modifiedmolecule does not have the same cysteine pattern as the standardmolecule folding would be affected. Another well known and acceptedmethod for measuring folding is circular dichrosism (CD). Measuring andcomparing the CD spectra generated by a modified molecule and standardmolecule would be routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known and accepted method for analyzingfolding and structure. Nuclear magnetic resonance (NMR), digestivepeptide mapping and epitope mapping are other known methods foranalyzing folding and structurally similarities between proteins andpolypeptides (Schaanan et al., Science 257:961-964, 1992).

[0076] A Hopp/Woods hydrophilicity profile of the zsig81 proteinsequence as shown in SEQ ID NO:2 can be generated (Hopp et al., Proc.Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. Hydrophilicity can beused to determine regions that have the most antigenic potential. Forexample, in zsig81, hydrophilic regions include amino acid residues101-107 of SEQ ID NO: 2, amino acid residues 57-62 of SEQ ID NO: 2,amino acid residues 17-22 of SEQ ID NO: 2, amino acid residues 16-21 ofSEQ ID NO: 2, and amino acid residues 100-105 of SEQ ID NO: 2.

[0077] Amino acid sequence changes are made in zsig81 polypeptides so asto minimize disruption of higher order structure essential to biologicalactivity. For example changes in amino acid residues will be made so asnot to disrupt the four-helix bundle characteristic of the proteinfamily. Methods for analyzing the effect of modification in primarysequence on secondary and tertiary structure are described herein. Thoseskilled in the art will recognize that hydrophilicity will be taken intoaccount when designing alterations in the amino acid sequence of azsig81 polypeptide, so as not to disrupt the overall profile. Residueswithin the core of the four-helix bindle can be replaced with a residueas shown in SEQ ID NO: 6, 7, 8, and 9. Of particular interest forreplacement are hydrophobic residues selected from the group consistingof Val, Leu and Ile or the group consisting of Met, Gly, Ser, Ala, Tyrand Trp. Cysteine residues at positions 2 and 76 of SEQ ID NO: 2, andthe residues predicted to be on the exposed surface of the four-helixbundle will be relatively intolerant of substitution.

[0078] Polypeptides of the present invention comprise at least 6,preferably at least 9, more preferably at least 15 contiguous amino acidresidues of SEQ ID NO:2. Within certain embodiments of the invention,the polypeptides comprise 20, 30, 40, 50, 100, or more contiguousresidues of SEQ ID NO:2, up to the entire mature polypeptide (residues Ito 156 of SEQ ID NO:2) or the primary translation product (residues −17to 156 of SEQ ID NO:2). As disclosed in more detail below, thesepolypeptides can further comprise additional, non-zsig81 polypeptidesequence(s).

[0079] Within the polypeptides of the present invention are polypeptidesthat comprise an epitope-bearing portion of a protein as shown in SEQ IDNO:2. An “epitope” is a region of a protein to which an antibody canbind. See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002, 1984. Epitopes can be linear or conformational, the latterbeing composed of discontinuous regions of the protein that form anepitope upon folding of the protein. Linear epitopes are generally atleast 6 amino acid residues in length. Relatively short syntheticpeptides that mimic part of a protein sequence are routinely capable ofeliciting an antiserum that reacts with the partially mimicked protein.See, Sutcliffe et al., Science 219:660-666, 1983. Antibodies thatrecognize short, linear epitopes are particularly useful in analytic anddiagnostic applications that employ denatured protein, such as Westernblotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979), or inthe analysis of fixed cells or tissue samples. Antibodies to linearepitopes are also useful for detecting fragments of zsig81 such as mightoccur in body fluids or cell culture media.

[0080] Antigenic, epitope-bearing polypeptides of the present inventionare useful for raising antibodies, including monoclonal antibodies, thatspecifically bind to a zsig81 protein. Antigenic, epitope-bearingpolypeptides contain a sequence of at least six, preferably at leastnine, more preferably from 15 to about 30 contiguous amino acid residuesof a zsig81 protein (e.g., SEQ ID NO:2). Polypeptides comprising alarger portion of a zsig81 protein, i.e. from 30 to 50 residues up tothe entire sequence, are included. It is preferred that the amino acidsequence of the epitope- bearing polypeptide is selected to providesubstantial solubility in aqueous solvents, that is the sequenceincludes relatively hydrophilic residues, and hydrophobic residues aresubstantially avoided. Preferred such regions include residues 16-21,17-22, 57-62, 100-105 and 101-107 of SEQ ID NO:2.

[0081] Using the methods discussed herein, one of ordinary skill in theart can identify and/or prepare a variety of polypeptides that are havesubstantially similar sequence identity to residues −17 to 156 or 1 to156 of SEQ ID NO: 2, or functional fragments, e.g., residues 5 to 156 ofSEQ ID NO: 2, and fusions thereof, and retain the properties of thewild-type protein such as the ability to stimulate proliferation,differentiation or induce specialized cell function.

[0082] Regardless of the particular nucleotide sequence of a variantzsig81 sequence, the sequence encodes a polypeptide that ischaracterized by its proliferative or differentiating activity, orability to induce specialized cell functions, or by the ability to bindspecifically to an anti-zsig81 antibody or antagonist that binds to thezsig81 receptor. More specifically, variant zsig81 genes encodepolypeptides which exhibit at least 50% and preferably, greater than 70,80 or 90%, of the activity of polypeptide encoded by the human zsig81gene described herein.

[0083] For any zsig81 polypeptide, including variants and fusionproteins, one of ordinary skill in the art can readily generate a fullydegenerate polynucleotide sequence encoding that variant using theinformation set forth in Tables 1 and 2 above.

[0084] The present invention further provides a variety of otherpolypeptide fusions (and related multimeric proteins comprising one ormore polypeptide fusions). For example, a zsig81 polypeptide can beprepared as a fusion to a dimerizing protein as disclosed in U.S. Pat.Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in thisregard include immunoglobulin constant region domains. Immunoglobulin-zsig81 polypeptide fusions can be expressed in genetically engineeredcells (to produce a variety of multimeric zsig81 analogs). Auxiliarydomains can be fused to zsig81 polypeptides to target them to specificcells, tissues, or macromolecules (e.g., collagen). For example, azsig81 polypeptide or protein could be targeted to a predetermined celltype by fusing a zsig81 polypeptide to a ligand that specifically bindsto a receptor on the surface of the target cell, or fused to an antibodydirected to an antigen expressed on a specific target cell type. In thisway, polypeptides and proteins can be targeted for therapeutic ordiagnostic purposes. A zsig81 polypeptide can be fused to two or moremoieties, such as an affinity tag for purification and a targetingdomain. Polypeptide fusions can also comprise one or more cleavagesites, particularly between domains. See, Tuan et al., Connective TissueResearch 34:1-9, 1996.

[0085] The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al., eds., Current Protocols in Molecular Biology, John Wileyand Sons, Inc., New York, 1987.

[0086] In general, a DNA sequence encoding a zsig81 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0087] To direct a zsig81 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the zsig81 polypeptide, ormay be derived from another secreted protein (e.g., t-PA) or synthesizedde novo. The secretory signal sequence is operably linked to the zsig81DNA sequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

[0088] Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residue −17to 1 of SEQ ID NO:2 is operably linked to a DNA sequence encodinganother polypeptide using methods known in the art and disclosed herein.The secretory signal sequence contained in the fusion polypeptides ofthe present invention is preferably fused amino-terminally to anadditional peptide to direct the additional peptide into the secretorypathway. Such constructs have numerous applications known in the art.For example, these novel secretory signal sequence fusion constructs candirect the secretion of an active component of a normally non-secretedprotein. Such fusions may be used in vivo or in vitro to direct peptidesthrough the secretory pathway.

[0089] Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993), and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

[0090] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

[0091] Other higher eukaryotic cells can also be used as hosts,including plant cells, insect cells and avian cells. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987. Transformation of insect cells and production of foreignpolypeptides therein is disclosed by Guarino et al., U.S. Pat. No.5,162,222 and WIPO publication WO 94/06463. Insect cells can be infectedwith recombinant baculovirus, commonly derived from Autographacalifornica nuclear polyhedrosis virus (AcNPV). DNA encoding the zsig81polypeptide is inserted into the baculoviral genome in place of theAcNPV polyhedrin gene coding sequence by one of two methods. The firstis the traditional method of homologous DNA recombination betweenwild-type AcNPV and a transfer vector containing the zsig81 flanked byAcNPV sequences. Suitable insect cells, e.g. SF9 cells, are infectedwith wild-type AcNPV and transfected with a transfer vector comprising azsig81 polynucleotide operably linked to an AcNPV polyhedrin genepromoter, terminator, and flanking sequences. See, King, L. A. andPossee, R. D., The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly, D. R. et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and, Richardson, C. D., Ed., Baculovirus Expression Protocols. Methodsin Molecular Biology, Totowa, N.J., Humana Press, 1995. Naturalrecombination within an insect cell will result in a recombinantbaculovirus which contains zsig81 driven by the polyhedrin promoter.Recombinant viral stocks are made by methods commonly used in the art.

[0092] The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, V. A, et al., JVirol 67:4566-79, 1993). This system is sold in the Bac-to-Bac kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBac1™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the zsig81 polypeptide into a baculovirus genome maintainedin E. coli as a large plasmid called a “bacmid.” The pFastBac1™ transfervector utilizes the AcNPV polyhedrin promoter to drive the expression ofthe gene of interest, in this case zsig81. However, pFastBac1™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkins, M. S. and Possee, R.D., J. Gen. Virol. 71:971-6, 1990; Bonning, B. C. et al., J. Gen. Virol.75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J. Biol.Chem. 270:1543-9, 1995. In such transfer vector constructs, a short orlong version of the basic protein promoter can be used. Moreover,transfer vectors can be constructed which replace the native zsig81secretory signal sequences with secretory signal sequences derived frominsect proteins. For example, a secretory signal sequence fromEcdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen,Carlsbad, Calif.), or baculovirus gp67 (PharMingen, San Diego, Calif.)can be used in constructs to replace the native zsig81 secretory signalsequence. In addition, transfer vectors can include an in-frame fusionwith DNA encoding an epitope tag at the C- or N-terminus of theexpressed zsig81 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Usinga technique known in the art, a transfer vector containing zsig81 istransformed into E. Coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses zsig81 is subsequentlyproduced. Recombinant viral stocks are made by methods commonly used theart.

[0093] The recombinant virus is used to infect host cells, typically acell line derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2-5×10⁵cells to a density of 1-2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. The recombinant virus-infected cells typically producethe recombinant zsig81 polypeptide at 12-72 hours post-infection andsecrete it with varying efficiency into the medium. The culture isusually harvested 48 hours post-infection. Centrifugation is used toseparate the cells from the medium (supernatant). The supernatantcontaining the zsig81 polypeptide is filtered through micropore filters,usually 0.45 μm pore size. Procedures used are generally described inavailable laboratory manuals (King, L. A. and Possee, R. D., ibid.;O'Reilly, D. R. et al., ibid.; Richardson, C. D., ibid.). Subsequentpurification of the zsig81 polypeptide from the supernatant can beachieved using methods described herein.

[0094] Fungal cells, including yeast cells, can also be used within thepresent invention. Yeast species of particular interest in this regardinclude Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Methods for transforming S. cerevisiae cells with exogenousDNA and producing recombinant polypeptides therefrom are disclosed by,for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S.Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S.Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075.Transformed cells are selected by phenotype determined by the selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient (e.g., leucine). A preferred vector system foruse in Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

[0095] The use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed in WIPO Publications WO 97/17450, WO97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use intransforming P. methanolica will commonly be prepared asdouble-stranded, circular plasmids, which are preferably linearizedprior to transformation. For polypeptide production in P. methanolica,it is preferred that the promoter and terminator in the plasmid be thatof a P. methanolica gene, such as a P. methanolica alcohol utilizationgene (AUG1 or AUG2). Other useful promoters include those of thedihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), andcatalase (CAT) genes. To facilitate integration of the DNA into the hostchromosome, it is preferred to have the entire expression segment of theplasmid flanked at both ends by host DNA sequences. A preferredselectable marker for use in Pichia methanolica is a P. methanolica ADE2gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;EC 4.1.1.21), which allows ade2 host cells to grow in the absence ofadenine. For large-scale, industrial processes where it is desirable tominimize the use of methanol, it is preferred to use host cells in whichboth methanol utilization genes (AUG1 and AUG2) are deleted. Forproduction of secreted proteins, host cells deficient in vacuolarprotease genes (PEP4 and PRB1) are preferred. Electroporation is used tofacilitate the introduction of a plasmid containing DNA encoding apolypeptide of interest into P. methanolica cells. It is preferred totransform P. methanolica cells by electroporation using an exponentiallydecaying, pulsed electric field having a field strength of from 2.5 to4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (ω) of from1 to 40 milliseconds, most preferably about 20 milliseconds.

[0096] Prokaryotic host cells, including strains of the bacteriaEscherichia coli, Bacillus and other genera are also useful host cellswithin the present invention. Techniques for transforming these hostsand expressing foreign DNA sequences cloned therein are well known inthe art (see, e.g., Sambrook et al., ibid.). When expressing a zsig81polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

[0097] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

[0098] Expressed recombinant zsig81 polypeptides (or chimeric zsig81polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers. Methods for bindingreceptor polypeptides to support media are well known in the art.Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

[0099] The polypeptides of the present invention can be isolated byexploitation of size, charge and hydrophobicity. For example,immobilized metal ion adsorption (IMAC) chromatography can be used topurify histidine-rich proteins (E. Sulkowski, Trends in Biochem. 3:1-7,1985). Other methods of purification include purification ofglycosylated proteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), Acad. Press, San Diego, 1990,pp.529-39). Within additional embodiments of the invention, a fusion ofthe polypeptide of interest and an affinity tag (e.g., maltose-bindingprotein, an immunoglobulin domain) may be constructed to facilitatepurification. ZSIG81 has a domain homologous to the heparin bindingdomain described previously for CTGF, and exploitation of this propertymay be useful for purification of zsig81. For a review, see, Burgess etal., Ann. Rev. of Biochem. 58:575-606, 1989. Members of the FGF family,which also have a heparin binding domain can be purified to apparenthomogeneity by heparin-Sepharose affinity chromatography (Gospodarowiczet al., Proc. Natl. Acad. Sci. 81:6963-6967, 1984) and eluted usinglinear step gradients of NaCl (Ron et al., J. Biol. Chem.268(4):2984-2988, 1993; Chromatography: Principles & Methods, pp. 77-80,Pharmacia LKB Biotechnology, Uppsala, Sweden, 1993; in “ImmobilizedAffinity Ligand Techniques”, Hermanson et al., eds., pp. 165-167,Academic Press, San Diego, 1992; Kjellen et al., Ann. Rev. Biochem.Ann.Rev. Biochem. 60:443-474, 1991; and Ke et al., Protein Expr. Purif.3(6):497-507, 1992.)

[0100] Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

[0101] Zsig81 polypeptides or fragments thereof may also be preparedthrough chemical synthesis (Merrifield, J. Am. Chem. Soc. 85:2149,1963). Zsig81 polypeptides may be monomers or multimers; glycosylated ornon-glycosylated; pegylated or non-pegylated; and may or may not includean initial methionine amino acid residue.

[0102] The activity of molecules of the present invention can bemeasured using a variety of assays that measure cell proliferation,differentiation, chemotaxis or induction of specialized cell functions.Of particular interest are changes in proliferation or differentiationof cells, particularly cells isolated from heart or liver tissue.Proliferation and differentiation can be measured ill vitro usingcultured cells or in vivo by administering molecules of the claimedinvention to the appropriate animal model. Assays measuring cellproliferation or differentiation are well known in the art. For example,assays measuring proliferation include such assays as chemosensitivityto neutral red dye (Cavanaugh et al., Investigational New Drugs8:347-354, 1990, incorporated herein by reference), incorporation ofradiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7,1989, incorporated herein by reference), incorporation of5-bromo-2′-deoxyuridine (BrdU) in the DNA of proliferating cells(Porstmann et al., J. Immunol. Methods 82:169-179, 1985, incorporatedherein by reference), and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589-601, 1988;Marshall et al., Growth Reg. 5:69-84, 1995; and Scudiero et al., CancerRes. 48:4827-4833, 1988; all incorporated herein by reference). Assaysmeasuring differentiation include, for example, measuring cell-surfacemarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference).

[0103] Examples of assays measuring induction of specialized cellfunctions include: extracellular matrix protein mRNA induction assays(Frazier et al., J. Invest. Dermatol. 107:404-411, 1996); ³⁵S methioninepulse-chase assays measuring stimulation of matrix protein synthesis(Frazier et al., ibid., 1996); subcutaneous administration of growthfactors to mice (Roberts et al., Proc. Natl. Acad. Sci. USA83:4167-4171, 1986); and in situ hybridization to measure changes inmRNA expression (Fava et al.,Blood 76:1946-1955, 1990).

[0104] Cell migration is assayed essentially as disclosed by Kahler etal. (Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939,1997). A protein is considered to be chemotactic if it induces migrationof cells from an area of low protein concentration to an area of highprotein concentration.

[0105] Cell adhesion activity is assayed essentially as disclosed byLaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly,microtiter plates are coated with the test protein, non-specific sitesare blocked with BSA, and cells (such as smooth muscle cells,leukocytes, or endothelial cells) are plated at a density ofapproximately 10⁴-10⁵ cells/well. The wells are incubated at 37° C.(typically for about 60 minutes), then non-adherent cells are removed bygentle washing. Adhered cells are quantitated by conventional methods(e.g., by staining with crystal violet, lysing the cells, anddetermining the optical density of the lysate). Control wells are coatedwith a known adhesive protein, such as fibronectin or vitronectin.

[0106] Assays for angiogenic activity are also known in the art. Forexample, the effect of zsig81 proteins on primordial endothelial cellsin angiogenesis can be assayed in the chick chorioallantoic membraneangiogenesis assay (Leung, Science 246:1306-1309, 1989; Ferrara, Ann. NYAcad. Sci. 752:246-256, 1995). Other suitable assays includemicroinjection of early stage quail (Coturnix cotunix japonica) embryosas disclosed by Drake et al. (Proc. Natl. Acad. Sci. USA 92:7657-7661,1995); the rodent model of corneal neovascularization disclosed byMuthukkaruppan and Auerbach (Science 205:1416-1418, 1979), wherein atest substance is inserted into a pocket in the cornea of an inbredmouse; and the hampster cheek pouch assay (Höckel et al., Arch. Surg.128:423-429, 1993).

[0107] The biological activities of zsig81 proteins can be studied innon-human animals by administration of exogenous protein, by expressionof zsig81-encoding polynucleotides, and by suppression of endogenouszsig81 expression through antisense or knock-out techniques. Zsig81proteins can be administered or expressed individually, in combinationwith other zsig81 proteins, or in combination with non-zsig81 proteins,including other growth factors. Test animals are monitored for changesin such parameters as clinical signs, body weight, blood cell counts,clinical chemistry, histopathology, and the like.

[0108] Stimulation of coronary collateral growth can be measured inknown animal models, including a rabbit model of peripheral limbischemia and hind limb ischemia and a pig model of chronic myocardialischemia (Ferrara et al., Endocrine Reviews 18:4-25, 1997). Zsig81proteins are assayed in the presence and absence of VEGFs,angiopoietins, and basic FGF to test for combinatorial effects. Thesemodels can be modified by the use of adenovirus or naked DNA for genedelivery as disclosed in more detail below, resulting in localexpression of the test protein(s).

[0109] Efficacy of zsig81polypeptides in promoting wound healing can beassayed in animal models. One such model is the linear skin incisionmodel of Mustoe et al. (Science 237:1333, 1987). Subcutaneous implantscan be used to assess compounds acting in the early stages of woundhealing (Broadley et al., Lab. Invest. 61:571, 1985; Sprugel et al.,Amer. J. Pathol. 129: 601, 1987).

[0110] Expression of zsig81 proteins in animals provides models forstudy of the biological effects of overproduction or inhibition ofprotein activity in vivo. Zsig81-encoding polynucleotides can beintroduced into test animals, such as mice, using viral vectors or nakedDNA, or transgenic animals can be produced. In general it is preferredto express a zsig81 protein with a secretory peptide. Suitable secretorypeptides include the zsig81 secretory peptide (e.g., residues −17 to 1of SEQ ID NO:2) and heterologous secretory peptides. A preferredheterologous secretory peptide is that of human tissue plasminogenactivator (t-PA). The t-PA secretory peptide may be modified to reduceundesired proteolytic cleavage as disclosed in U.S. Pat. No. 5,641,655.

[0111] One in vivo approach for assaying proteins of the presentinvention utilizes viral delivery systems. Exemplary viruses for thispurpose include adenovirus, herpesvirus, retroviruses, vaccinia virus,and adeno-associated virus (AAV). Adenovirus, a double-stranded DNAvirus, is currently the best studied gene transfer vector for deliveryof heterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

[0112] Using adenovirus vectors where portions of the adenovirus genomeare deleted, inserts are incorporated into the viral DNA by directligation or by homologous recombination with a co-transfected plasmid.In an exemplary system, the essential E1 gene has been deleted from theviral vector, and the virus will not replicate unless the E1 gene isprovided by the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined. Adenoviralvectors containing various deletions of viral genes can be used in anattempt to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1 deleted, and in addition contain deletions of E2A orE4 (Lusky et al., J. Virol. 72:2022-2032, 1998; Raper et al., Human GeneTherapy 9:671-679, 1998). In addition, deletion of E2b is reported toreduce immune responses (Amalfitano, et al., J. Virol. 72:926-933,1998). Generation of so-called “gutless” adenoviruses where all viraltranscription units are deleted is particularly advantageous forinsertion of large inserts of heterologous DNA. For review, see Yeh andPerricaudet, FASEB J. 11:615-623, 1997.

[0113] In another embodiment, a zsig81 gene can be introduced in aretroviral vector as described, for example, by Anderson et al., U.S.Pat. No. 5,399,346; Mann et al., Cell 33:153, 1983; Temin et al., U.S.Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz etal., J. Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;Dougherty et al., WIPO publication WO 95/07358; and Kuo et al., Blood82:845, 1993.

[0114] In an alternative method, the vector can be introduced by“lipofection” in vivo using liposomes. Synthetic cationic lipids can beused to prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987;Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use oflipofection to introduce exogenous genes into specific organs in vivohas certain practical advantages. Molecular targeting of liposomes tospecific cells represents one area of benefit. For instance, directingtransfection to particular cell types is particularly advantageous in atissue with cellular heterogeneity, such as the pancreas, liver, kidney,and brain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

[0115] Within another embodiment target cells are removed from the theanimal, and the DNA is introduced as a naked DNA plasmid. Thetransformed cells are then re-implanted into the body of the animal.Naked DNA vectors can be introduced into the desired host cells bymethods known in the art, e.g., transfection, electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, use of a gene gun or use of a DNA vectortransporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992; Wu etal., J. Biol. Chem. 263:14621-4, 1988.

[0116] Mice engineered to express the zsig81 gene, referred to as“transgenic mice,” and mice that exhibit a complete absence of zsig81gene function, referred to as “knockout mice,” can also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, Science 244:1288-1292, 1989; Palmiter etal., Ann. Rev. Genet. 20:465-499, 1986). Transgenesis experiments can beperformed using normal mice or mice with genetic disease or otheraltered phenotypes. Transgenic mice that over-express zsig81, eitherubiquitously or under a tissue-specific or tissue-restricted promoter,can be used to determine whether or not over-expression causes aphenotypic change. Preferred promoters include metallothionein andalbumin gene promoters. The metallothionein-1 (MT-1) promoter providesexpression in liver and other tissues, often leading to high levels ofcirculating protein. Over-expression of a wild-type zsig81 polypeptide,polypeptide fragment or a mutant thereof may alter normal cellularprocesses, resulting in a phenotype that identifies a tissue in whichzsig81expression is functionally relevant and may indicate a therapeutictarget for the zsig81, its agonists or antagonists. For example, apreferred transgenic mouse to engineer is one that over-expresses afull-length zsig81 sequence. Such over-expression may result in aphenotype that shows similarity with human diseases. Similarly, knockoutzsig81 mice can be used to determine where zsig81 is absolutely requiredin vivo. The phenotype of knockout mice is predictive of the in vivoeffects of zsig81antagonists. Knockout mice can also be used to studythe effects of zsig81 proteins in models of disease, including, forexample, cancer, atherosclerosis, rheumatoid arthritis, ischemia, andcardiovascular disease. The human zsig81 cDNA can be used to isolatemurine zsig81 mRNA, cDNA and genomic DNA as disclosed above, which aresubsequently used to generate knockout mice. These mice may be employedto study the zsig81 gene and the protein encoded thereby in an in vivosystem, and can be used as in vivo models for corresponding humandiseases. Moreover, transgenic mice expressing zsig81 antisensepolynucleotides or ribozymes directed against zsig81, described herein,can be used analogously to knockout mice described above.

[0117] Antisense methodology can be used to inhibit zsig81 genetranscription to examine the effects of such inhibition in vivo.Polynucleotides that are complementary to a segment of a zsig81-encodingpolynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:1) aredesigned to bind to zsig81-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense oligonucleotides can also be used to inhibitexpression of zsig81 polypeptide-encoding genes in cell culture.

[0118] Proteins of the present invention are useful for modulating theproliferation, differentiation, migration, or metabolism of responsivecell types, which include both primary cells and cultured cell lines. Ofparticular interest in this regard are hematopoietic cells (includingstem cells and mature myeloid and lymphoid cells), endothelial cells,and mesenchymal cells (including fibroblasts, and cardiac and smoothmuscle cells). Zsig81 polypeptides are added to tissue culture media forthese cell types at a concentration of about 10 pg/ml to about 1000ng/ml. Those skilled in the art will recognize that zsig81 proteins canbe advantageously combined with other growth factors in culture media.

[0119] Dendritic cells are the most potent antigen presenting cells(APCs) in the immune system. Dendritic cells are the only cells thatpresent antigen to, and activate, naive CD4⁺ T cells in vivo (Levin etal., J. Immunol. 151:6742-6750, 1993). Dendritic cells are found inprimary and secondary lymphoid organs (e.g., thymus, lymph nodes,tonsils, Peyer's patches, and spleen), as well as in non-lymphoid organsand tissues (e.g., heart, liver, lung, gut, and in the skin as epidermalLangerhans cells). Dendritic cells are also prevalent in afferent lymph,but are rare in blood. For reviews, see Steinman, Ann. Rev. Immunol.9:271-296, 1991 and Knight et al., J. Invest. Dermatol. 99:33S-38S,1992.

[0120] Dendritic cells are thought to originate from a singlehematopoietic progenitor cell. As progenitor cells begin the process ofdifferentiation they migrate to selected tissue and/or organs, wherethey appear to undergo additional differentiation. If isolated fromtissue, dendritic cells are immature; that is, the cells are not fullydifferentiated, are inefficient at antigen presentation, express lowlevels of MHC Class II molecules and do not stimulate proliferation ofT-cells in an allogenic mixed leukocyte reaction (MLR). However, whenimmature dendritic cells are exposed to foreign proteins, they becomecapable of taking up and presenting soluble antigen via newlysynthesized MHC Class II molecules, and simultaneously leave theirtissue residence and migrate to lymph nodes and spleen. After migratingfrom the origin tissue, the dendritic cells are mature; that is, theyexhibit high levels of MHC Class II, accessory and co-stimulatorymolecules, as well as full APC function (Steinman, ibid., 1990 andIbrahim et al., Immunol. Today 16:181-186, 1995). Recently, availabilityof immortalized dendritic cells have expanded scientists' ability toinvestigate antigen uptake and processing by dendritic cells and theirprecursor cells (see, e.g., U.S. Pat. No. 5,648,219.)

[0121] Dendritic cells have been implicated as a causative cell-type ina number of different diseases that involve immune responses, includingcontact sensitivity, tumor immunity, HIV-1 infection and autoimmunity(e.g., Type I diabetes, multiple sclerosis and rheumatoid arthritis).These cells are believed to play a role in graft rejection, where cellsfrom the allograft migrate into the lymphoid organs of the recipient andinitiate a deleterious immune response.

[0122] Recent investigations have demonstrated that cytokines play anessential role in the maturation and ability of dendritic cells topresent antigen. GM-CSF was found to enhance antigen presentation byLangerhans cells in vivo and tumor immunity in vitro in other dendriticcells (Grabbe et al., Immunology Today 16:117-121, 1995.) Othercytokines believed to play a role in dendritic cell function anddifferentiation include: FLT3 ligand, IL-12, KIT ligand, TNT-α and IL-4in conjunction with GM-CSF (Shurin et al., Cytokine & Growth FactorReviews 9:37-48, 1998); Stem cell factor, TGF-β, IL-6 and IL-3 (Bruggeret al., Annals N.Y. Acad. Sci. 872:363-371, 1999).

[0123] Molecules of the present invention have been shown to stimulatethe proliferation of cells expressing markers associated with dendriticlineage cells. Conditioned medium from human embryonic kidney cellstransfected with cDNA for murine zsig81 stimulated proliferation of bonemarrow cultures. The bone marrow culture contained multiple celllineages, at various stages of differentiation, and the outgrowth fromthe cultures resulted in a significant increase in cells that wereCD80+, CD86+, MHC II and CD11c⁺, which are markers for dendritic cellsof a possible lymphoid or myeloid origin. The identification of thymiclymphoid-related dendritic cells are phenotypically distinguishable frommyeloid related dendritic cells. While the various functionaldifferences between dendritic cells of lymphoid and myeloid origin havenot been fully elucidated, lymphoid related dendritic cells may beinvolved in self-antigen tolerance, while myeloid related dendriticcells involved in endocytosis of foreign antigens (de St. Groth,Immunology Today 19:448-454, 1998.)

[0124] Dendritic cells have activities that are specifically associatedwith the maturity of the cell, i.e., its differentiated state. Toidentify a cell's maturity, a population of established cells is assayedand analyzed for a set of differentiation markers that arecharacteristic of the cell's stage in the differentiation pathway.Preferably, this is done by isolating at least a portion of the cellsand subjecting this subpopulation to such analysis.

[0125] A set of differentiation markers is defined as one or morephenotypic properties that can be identified, and that are specific to aparticular cell type and stage of maturity. Differentiation markers aretransiently exhibited at various stages of the cell's progression towardterminal differentiation. Pluripotent stem cells that can regeneratewithout commitment to a specific cell lineage express a set ofdifferentiation markers that are diminished when commitment to aparticular cell lineage is made. Precursor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsusually represent functional properties, such as cell products, enzymesto produce cell products and receptors. It is possible that withexposure to the appropriate factors, the cell line of the presentinvention can differentiate and mature into other cells of the monocyticcell lineage. Differentiation markers used for identifying dendriticcells include: Mac-1, F4/80, FcγRII/III receptor (FcR), MHC class I, MHCclass II, B7-1, B7-2, ICAM-1, CD44, N418, and NLDC-145.

[0126] In immature dendritic cells, F4/80 (Lee et al., J. Exp. Med.161:475, 1985) and FcR (Unkeless, J. Exp. Med. 150:580, 1979) aredetectable, but at levels lower than those seen in a macrophage usingmonoclonal antibodies that bind F4/80 (Caltag, San Fransisco, Calif.)and 2.4G2 for FcR binding (PharMingen, San Diego, Calif.); MHC class Iis detectable using the monoclonal antibody EH144.3 (Geier et al., J.Immunol. 137:1239, 1986); MHC class II is detectable only at low levelsusing the monoclonal antibody AF6-120.1 (PharMingen); B7-1 and B7-2 aredetectable at low levels (Nabavi et al., Nature 360:266, 1992 andHathcock et al., Science 262:905, 1993, respectively) using monoclonalantibodies IG10 (PharMingen) and GL1 (PharMingen); ICAM-1 (Rothlein etal., J. Immunol. 137:1270, 1986), using monoclonal antibody 3E2(PharMingen), and CD44 (Lesley et al., Immunogenetics 15:313, 1982),using monoclonal antibody IM7 (PharMingen), are detectable at highlevels; and at least one of the dendritic cell markers CD11c (Metaly etal., J. Exp. Med. 171:1753, 1990), using the monoclonal antibody N418,or DC-205 (Kraal et al., J. Exp. Med. 163:981, 1986), using themonoclonal antibodies NLDC-145 (Accurate Chem. and Scientific, Westbury,N.Y.) and 33D1 (Nussenzweig et al., Proc. Natl. Acad. Sci. USA. 79:161,1982), should be detectable. The skilled practitioner would recognizethat not all of these differentiation markers may be present and thatexpression levels may vary. In activated dendritic cells, high levels ofMHC class II are detectable; B7-2 and ICAM-1 are expressed at higherlevels, and F4/80 is expressed at lower levels than seen in immaturedendritic cells.

[0127] Analyses of the cell surface using monoclonal antibodies are madeusing a flow cytometer, see, for example, Fink et al., J. Exp. Med.176:1733, 1992 and Crowley et al., Cellular Immunol. 118:108-125, 1989.Briefly, the cells are either combined with monoclonal antibodiesdirectly conjugated to fluorochromes, or with unconjugated primaryantibody and subsequently with commercially available secondaryantibodies conjugated to fluorochromes. The stained cells are analyzedusing a FACScan (Becton Dickinson, Mountain View, Calif.) using LYSYS IIor Cell Quest software (Becton Dickinson).

[0128] Identification of activated dendritic cells is confirmed by thecells' ability to stimulate the proliferation of allogeneic T cells in aMLR. Briefly, activated dendritic cells are incubated with allogeneic Tcells in a 96-well microtiter dish (American Scientific Products,Chicago, Ill.). Stimulation of the T cells to proliferate is measured byincorporation of ³H-thymidine. It is preferred to expose the dendriticcells of the present invention to irradiation to slow the proliferationof the dendritic cells and reduce background in the assay caused byincorporation of ³H-thymidine by the dendritic cells.

[0129] The dendritic cells are activated to induce expression of MHCclass II molecules on the cell surface, making these mature dendriticcells competent for antigen processing and presentation. These activatedcells (i.e., stimulators) are then exposed to antigen for a timesufficient for antigen presentation. One skilled in the art wouldrecognize that the time required for endocytosis, processing andpresentation of antigen is dependent upon the proteinaceous antigenbeing used for this purpose. Methods for measuring antigen uptake andpresentation are known in the art. For example, dendritic cells can beincubated with a soluble protein antigen (e.g., ovalbumin or conalbumin)for 3-24 hours then washed to remove exogenous antigen.

[0130] These antigen-presenting stimulator cells are then mixed withresponder cells, preferably naive or antigen-primed T lymphocytes. Afteran approximately 72 hour incubation (for primed T lymphocytes) orapproximately 4-7 d period (for naive T lymphocytes), the activation ofT cells in response to the processed and presented antigen is measured.In a preferred embodiment, T cell activation is determined by measuringT cell proliferation using ³H-thymidine uptake (Crowley et al., J.Immunol. Meth. 133:55-66, 1990). The responder cells in this regard canbe PBMN cells, cultured T cells, established T cell lines or hybridomas.Responder cell activation can be measured by the production ofcytokines, such as IL-2, or by determining T cell-specific activationmarkers. Cytokine production can be assayed by the testing the abilityof the stimulator+responder cell culture supernatant to stimulate growthof cytokine-dependent cells. T cell-specific activation markers may bedetected using antibodies specific for such markers.

[0131] For T cell proliferation assays, it is preferred to inhibit theproliferation of dendritic cells prior to mixing with T responder cells.This inhibition may be achieved by exposure to gamma irradiation or toan anti-mitotic agent, such as mitomycin C.

[0132] Alternatively, activated dendritic cells can be used to inducenon-responsiveness in T lymphocytes. In addition to MHC class IIrecognition, T cell activation requires co-receptors on theantigen-presenting cell (APC; e.g., the dendritic cell) that have beenstimulated with co-stimulatory molecules. By blocking or eliminatingstimulation of such co-receptors (for instance, by blocking withanti-receptor or anti-ligand antibodies, or by “knocking out” thegene(s) encoding such receptors), presentation of antigen byco-receptor-deficient dendritic cells can be used to render Tlymphocytes non-responsive to antigen.

[0133] Zsig81 proteins may be used either alone or in combination withother hematopoietic factors such as IL-3, G-CSF, GM-CSF, IL-4, M-CSF,IL-12 or stem cell factor to enhance expansion and mobilization ofhematopoietic or mesenchymal stem cells, including precursor stem cells.Cells that can be expanded in this manner include cells isolated frombone marrow, cells isolated from blood, neonatal heart or liver. Zsig81proteins may also be given directly to an individual to enhance stemcell production and differentiation within the treated individual. Inparticular, zsig81 can be used for expansion of dendritic cell anddendritic cell precursor populations.

[0134] Zsig81 proteins can also be used to identify inhibitors of theiractivity. Test compounds are added to the assays disclosed above toidentify compounds that inhibit the activity of zsig81 protein. Inaddition to those assays disclosed above, samples can be tested forinhibition of zsig81 activity within a variety of assays designed tomeasure receptor binding or the stimulation/inhibition ofzsig81-dependent cellular responses. For example, zsig81-responsive celllines can be transfected with a reporter gene construct that isresponsive to a zsig81-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a zsig81-activated serum response element (SRE) operably linkedto a gene encoding an assayable protein, such as luciferase. Candidatecompounds, solutions, mixtures or extracts are tested for the ability toinhibit the activity of zsig81 on the target cells as evidenced by adecrease in zsig81 stimulation of reporter gene expression. Assays ofthis type will detect compounds that directly block zsig81 binding tocell-surface receptors, as well as compounds that block processes in thecellular pathway subsequent to receptor-ligand binding. In thealternative, compounds or other samples can be tested for directblocking of zsig81 binding to receptor using zsig81 tagged with adetectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, andthe like). Within assays of this type, the ability of a test sample toinhibit the binding of labeled zsig81 to the receptor is indicative ofinhibitory activity, which can be confirmed through secondary assays.Receptors used within binding assays may be cellular receptors orisolated, immobilized receptors.

[0135] The activity of zsig81 proteins can be measured with asilicon-based biosensor microphysiometer that measures the extracellularacidification rate or proton excretion associated with receptor bindingand subsequent physiologic cellular responses. An exemplary such deviceis the Cytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif.. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including zsig81 proteins, their agonists, and antagonists.Preferably, the microphysiometer is used to measure responses of azsig81-responsive eukaryotic cell, compared to a control eukaryotic cellthat does not respond to zsig81 polypeptide. Zsig81-responsiveeukaryotic cells comprise cells into which a receptor for zsig81 hasbeen transfected creating a cell that is responsive to zsig81, as wellas cells naturally responsive to zsig81 such as cells derived fromvascular, cardiac, hematopoietic, or hepatic tissue. Differences,measured by a change, for example, an increase or diminution inextracellular acidification, in the response of cells exposed to zsig81polypeptide, relative to a control not exposed to zsig81, are a directmeasurement of zsig81-modulated cellular responses. Moreover, suchzsig81-modulated responses can be assayed under a variety of stimuli.The present invention thus provides methods of identifying agonists andantagonists of zsig81 proteins, comprising providing cells responsive toa zsig81 polypeptide, culturing a first portion of the cells in theabsence of a test compound, culturing a second portion of the cells inthe presence of a test compound, and detecting a change, for example, anincrease or diminution, in a cellular response of the second portion ofthe cells as compared to the first portion of the cells. The change incellular response is shown as a measurable change in extracellularacidification rate. Culturing a third portion of the cells in thepresence of a zsig81 protein and the absence of a test compound providesa positive control for the zsig81-responsive cells and a control tocompare the agonist activity of a test compound with that of the zsig81polypeptide. Antagonists of zsig81 can be identified by exposing thecells to zsig81 protein in the presence and absence of the testcompound, whereby a reduction in zsig81-stimulated activity isindicative of antagonist activity in the test compound.

[0136] Zsig81 proteins can also be used to identify cells, tissues, orcell lines that respond to a zsig81-stimulated pathway. Themicrophysiometer, described above, can be used to rapidly identifyligand-responsive cells, such as cells responsive to zsig81 proteins.Cells are cultured in the presence or absence of zsig81 polypeptide.Those cells that elicit a measurable change in extracellularacidification in the presence of zsig81 are responsive to zsig81.Responsive cells can than be used to identify antagonists and agonistsof zsig81 polypeptide as described above.

[0137] Inhibitors of zsig81 activity (zsig81 antagonists) includeanti-zsig81 antibodies and soluble zsig81 receptors, as well as otherpeptidic and non-peptidic agents, including ribozymes, small moleculeinhibitors, and angiogenically or mitogenically inactivereceptor-binding fragments of zsig81 polypeptides. Such antagonists canbe use to block biological activities of zsig81, including mitogenic,chemotactic, or angiogenic effects.

[0138] The polypeptides, nucleic acids, and antibodies of the presentinvention may be used in diagnosis or treatment of disorders associatedwith cell loss or abnormal cell proliferation (including cancer),including impaired or excessive vasculogenesis or angiogenesis andautoimmune disease. Labeled zsig81 polypeptides may be used for imagingtumors or other sites of abnormal cell proliferation.

[0139] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, and rats with a zsig81 polypeptide or a fragment thereof.

[0140] The immunogenicity of a zsig81 polypeptide may be increasedthrough the use of an adjuvant, such as alum (aluminum hydroxide) orFreund's complete or incomplete adjuvant. Polypeptides useful forimmunization also include fusion polypeptides, such as fusions of zsig81or a portion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

[0141] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

[0142] Antibodies are considered to be specifically binding if: 1) theyexhibit a threshold level of binding activity, and 2) they do notsignificantly cross-react with related polypeptide molecules. Athreshold level of binding is determined if anti-zsig81 antibodiesherein bind to a zsig81 polypeptide, peptide or epitope with an affinityat least 10-fold greater than the binding affinity to control(non-zsig81) polypeptide. It is preferred that the antibodies exhibit abinding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672,1949).

[0143] Whether anti-zsig81 antibodies do not significantly cross-reactwith related polypeptide molecules is shown, for example, by theantibody detecting zsig81 polypeptide but not known related polypeptidesusing a standard Western blot analysis (Ausubel et al., ibid.). Examplesof known related polypeptides are those disclosed in the prior art, suchas known orthologs, and paralogs. Screening can also be done usingnon-human zsig81, and zsig81 mutant polypeptides. Moreover, antibodiescan be “screened against” known related polypeptides, to isolate apopulation that specifically binds to the zsig81 polypeptides. Forexample, antibodies raised to zsig81 are adsorbed to relatedpolypeptides adhered to insoluble matrix; antibodies specific to zsig81will flow through the matrix under the proper buffer conditions.Screening allows isolation of polyclonal and monoclonal antibodiesnon-crossreactive to known closely related polypeptides (Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995).Screening and isolation of specific antibodies is well known in the art.See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff etal., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principlesand Practice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjaminet al., Ann. Rev. Immunol. 2: 67-101, 1984. Specifically bindinganti-zsig81 antibodies can be detected by a number of methods in theart, and disclosed below.

[0144] A variety of assays known to those skilled in the art can beutilized to detect antibodies which bind to zsig81 proteins orpolypeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant zsig81polypeptide.

[0145] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to zsig81 proteinor peptide, and selection of antibody display libraries in phage orsimilar vectors (for instance, through use of immobilized or labeledzsig81 protein or peptide). Genes encoding polypeptides having potentialzsig81 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the zsig81sequences disclosed herein to identify proteins which bind to zsig81.These “binding polypeptides” which interact with zsig81 polypeptides canbe used for tagging cells; for isolating homolog polypeptides byaffinity purification; they can be directly or indirectly conjugated todrugs, toxins, radionuclides and the like. These binding polypeptidescan also be used in analytical methods such as for screening expressionlibraries and neutralizing activity, e.g., for blocking interactionbetween ligand and receptor, or viral binding to a receptor. The bindingpolypeptides can also be used for diagnostic assays for determiningcirculating levels of zsig81 polypeptides; for detecting or quantitatingsoluble zsig81 polypeptides as marker of underlying pathology ordisease. These binding polypeptides can also act as zsig81 “antagonists”to block zsig81 binding and signal transduction in vitro and in vivo.These anti-zsig81 binding polypeptides would be useful for inhibitingzsig81 activity or protein-binding.

[0146] Antibodies to zsig81 may be used for tagging cells that expresszsig81; for isolating zsig81 by affinity purification; for diagnosticassays for determining circulating levels of zsig81 polypeptides; fordetecting or quantitating soluble zsig81 as marker of underlyingpathology or disease; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzsig81 in vitro and in vivo.

[0147] Antibodies or polypeptides herein may also be directly orindirectly conjugated to drugs, toxins, radionuclides, enzymes, and thelike, and these conjugates used for in vivo diagnostic or therapeuticapplications. Moreover, antibodies to zsig81 or fragments thereof may beused in vitro to detect denatured zsig81 or fragments thereof in assays,for example, Western Blots or other assays known in the art.

[0148] Antibodies or polypeptides herein can also be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (receptor orantigen, respectively, for instance). More specifically, zsig81polypeptides or anti-zsig81 antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues or organs that expressthe anti-complementary molecule.

[0149] Suitable detectable molecules may be directly or indirectlyattached to the polypeptide or antibody, and include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, saporinand the like), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Polypeptides or antibodies may also be conjugated tocytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the polypeptide or antibodyportion. For these purposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair.

[0150] In another embodiment, polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat diseases caused byinappropriate growth of cells or tissues). Such molecule fusion proteinsthus represent a generic targeting vehicle for cell/tissue-specificdelivery of generic anti-complementary-detectable/cytotoxic moleculeconjugates.

[0151] Molecules of the present invention can be used to identify andisolate receptors involved in growth and differentiation of zsig81responsive cells. For example, proteins and peptides of the presentinvention can be immobilized on a column and membrane preparations runover the column (Immobilized Affinity Ligand Techniques, Hermanson etal., eds., Academic Press, San Diego, Calif., 1992, pp.195-202).Proteins and peptides can also be radiolabeled (Methods in Enzymol.,vol. 182, “Guide to Protein Purification”, M. Deutscher, ed., Acad.Press, San Diego, 1990, 721-737) fluorescent or photoaffinity labeled(Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al.,Biochem. Pharmacol. 33:1167-1180, 1984) and specific cell-surfaceproteins can be identified.

[0152] Polynucleotides encoding zsig81 polypeptides are useful withingene therapy applications where it is desired to increase or inhibitzsig81 activity. If a mammal has a mutated or absent zsig81 gene, thezsig81 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zsig81 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-28, 1989).

[0153] In another embodiment, the zsig81 gene can be introduced in aretroviral vector, e.g., as described in Anderson et al., U.S. Pat. No.5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263;International Patent Publication No. WO 95/07358, published Mar. 16,1995 by Dougherty et al.; and Kuo et al., Blood 82:845-852, 1993.Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Natl. Acad. Sci. USA 84:7413-17, 1987; Mackey et al., Proc. Natl.Acad. Sci. USA 85:8027-31, 1988). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. More particularly, directingtransfection to particular cells represents one area of benefit. Forinstance, directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

[0154] It is possible to remove the target cells from the body; tointroduce the vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-67, 1992; Wu et al., J. Biol. Chem. 263:14621-24, 1988.

[0155] Antisense methodology can be used to inhibit zsig81 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of a zsig81-encodingpolynucleotide (e.g., a polynucleotide as set forth in SEQ ID NO:1) aredesigned to bind to zsig81-encoding mRNA and to inhibit translation ofsuch mRNA. Such antisense polynucleotides are used to inhibit expressionof zsig81 polypeptide-encoding genes in cell culture or in a subject.

[0156] For pharmaceutical use, the proteins of the present invention areformulated for parenteral, particularly intravenous or subcutaneous,delivery according to conventional methods. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. In general, pharmaceutical formulations will include azsig81 protein in combination with a pharmaceutically acceptablevehicle, such as saline, buffered saline, 5% dextrose in water or thelike. Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc. Methods of formulation are wellknown in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally be in therange of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20μg/kg per day, with the exact dose determined by the clinician accordingto accepted standards, taking into account the nature and severity ofthe condition to be treated, patient traits, etc. Determination of doseis within the level of ordinary skill in the art. The proteins may beadministered for acute treatment, over one week or less, often over aperiod of one to three days or may be used in chronic treatment, overseveral months or years. The invention is further illustrated by thefollowing non-limiting examples.

[0157] Thus, in summary, the certain embodiments of the presentinvention include an isolated polypeptide comprising at least ninecontiguous amino acid residues of SEQ ID NO: 2; as well as an isolatedpolypeptide comprising a sequence of amino acid residues selected fromthe group consisting of: (a) residues 30-44 of SEQ ID NO: 2; (b)residues 56-70 of SEQ ID NO: 2; (c) residues 86-94 of SEQ ID NO: 2; and(d) residues 135-149 of SEQ ID NO: 2. Furthermore, the polypeptide maycomprise residues 80-94 or 86-100 of SEQ ID NO: 2.

[0158] The isolated polypeptide of the present may also include asequence of amino acid residues that is at least 90% identical to aminoacid residues 30 to 149 of SEQ ID NO: 2, but also includes a truncatedmolecule comprising the sequence of amino acid residues comprisesresidues 5. to 156 of SEQ ID NO: 2. Further embodiments include themature polypeptide which comprises the sequence of amino acid residuescomprises residues 1 to 156 of SEQ ID NO: 2 and the primary translationproduct which comprises residues −17 to 156 of SEQ ID NO: 2.

[0159] In other embodiments, the present invention includes a fusionprotein comprising at least two polypeptides, wherein a firstpolypeptide is selected from the group consisting of: (a) residues 30-44of SEQ ID NO: 2; (b) residues 56-70 of SEQ ID NO: 2; (c) residues 86-94of SEQ ID NO: 2; and (d) residues 135-149 of SEQ ID NO: 2. In otheraspects, the a second polypeptide of the fusion protein is selected froma functional fragment of another cytokine, an antibody, or a toxinconjugate. In another aspect, the fusion protein comprising a firstpolypeptide and a second polypeptide, joined by a peptide bond, saidfirst polypeptide comprises a signal sequence and a second polypeptidecomprising an a sequence of amino acids as shown in SEQ ID NO: 2 fromamino acid residues 30-149. Other embodiments include a fusion proteincomprising a first polypeptide and a second polypeptide, joined by apeptide bond, wherein the first polypeptide is a maltose bindingprotein, the peptide bond is selected from the group consisting ofFactor Xa cleavage site, thrombin cleavage site or enterokinase cleavagesite, and the second polypeptide comprising an a sequence of amino acidsas shown in SEQ ID NO: 2 from amino acid residues 30-149.

[0160] In another embodiment, the present invention includes compositioncomprising a sequence of amino acid residues selected from the groupconsisting of: (a) residues 30-44 of SEQ ID NO: 2; (b) residues 56-70 ofSEQ ID NO: 2; (c) residues 86-94 of SEQ ID NO: 2; and (d) residues135-149 of SEQ ID NO: 2; and a pharmaceutically acceptable vehicle.

[0161] Also included in the present invention are embodiments directedto an isolated polynucleotide encoding a polypeptide comprising: (a)residues 30-44 of SEQ ID NO: 2; (b) residues 56-70 of SEQ ID NO: 2; (c)residues 86-94 of SEQ ID NO: 2; or (d) residues 135-149 of SEQ ID NO: 2.In another aspect, the present invention includes an isolatedpolynucleotide comprising: (a) nucleotides 272-316 of SEQ ID NO: 1; (b)nucleotides 350-394 of SEQ ID NO: 1; (c) nucleotides 440-466 of SEQ IDNO: 1; or (d) nucleotides 587-631 of SEQ ID NO: 1; and a polynucleotidecomprising: (a) nucleotides 139-183 of SEQ ID NO: 5; (b) nucleotides216-261 of SEQ ID NO: 5; (c) nucleotides 307-331 of SEQ ID NO: 5; or (d)nucleotides 454-499 of SEQ ID NO: 5. Another embodiment of the presentinvention includes an isolated polynucleotide encoding a polypeptidecomprising a sequence of amino acid residues that is at least 90%identical to amino acid residues 30 to 149 of SEQ ID NO: 2

[0162] In another embodiment, the present invention includes expressionvectors comprising the zsig81 polynucleotides described herein, andcultured cells containing those expression vectors.

[0163] Also included is a method of making a zsig 81 polypeptidecomprising: culturing a cell expressing zsig81 under conditions wherebythe DNA segment is expressed and the polypeptide is produced; andrecovering the polypeptide.

[0164] Other embodiments include an antibody which specifically binds toa zsig81 polypeptide.

[0165] Other embodiments of the present invention include a method forexpansion of hematopoietic cells and hematopoietic cell progenitorscomprising culturing bone marrow or peripheral blood cells with acomposition comprising an amount of zsig81 polypeptide sufficient toproduce an increase in the number of hematopoietic cells in the bonemarrow or peripheral blood cells as compared to bone marrow orperipheral blood cells cultured in the absence of zsig81. In addition,the hematopoietic cells and hematopoietic cell progenitors can belymphoid or myeloid cells. Of particular interest are hematopoieticcells and hematopoietic progenitor cells that are dendritic cells.

[0166] In another embodiment, the present invention includes a method ofmodulating an immune response in a mammal exposed to an antigencomprising: (1) determining a level of antigen-specific antibody; (2)administering a composition comprising zsig81 polypeptide in apharmaceutically acceptable vehicle; (3) determining a postadministration level of antigen-specific antibody; (4) comparing thelevel of antibody in step (1) to the level of antibody in step (3),wherein a change in antibody level is indicative of modulating theimmune response.

[0167] Another embodiment includes a method of detecting the presence ofzsig81 RNA in a biological sample, comprising the steps of: (a)contacting a zsig81 nucleic acid probe under hybridizing conditions witheither (i) test RNA molecules isolated from the biological sample, or(ii) nucleic acid molecules synthesized from the isolated RNA molecules,wherein the probe has a nucleotide sequence of nucleic acid molecule ofclaim 20, or its complement; and (b) detecting the formation of hybridsof the nucleic acid probe and either the test RNA molecules or thesynthesized nucleic acid molecules, wherein the presence of the hybridsindicates the presence of zsig81 in the biological sample.

[0168] Another embodiment of the present invention includes a method ofdetecting the presence of zsig81 in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody, or anantibody fragment, of claim 24, wherein the contacting is performedunder conditions that allow the binding of the antibody or antibodyfragment to the biological sample; and (b) detecting any of the boundantibody or bound antibody fragment.

[0169] In another embodiment, the present invention includes a methodfor stimulating antigenic response to tumor antigens comprising thesteps of: (1) isolating hematopoietic cells from a mammal; (2) exposingthe isolated hematopoietic cells to a tumor antigen; (3) culturing theexposed cells in a composition comprising an isolated polypeptide of atleast nine contiguous amino acid residues of SEQ ID NO: 2; and (4)administering the cultured cells back to the mammal.

[0170] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Northern Analysis of zsig81

[0171] Northern analyses were performed using Human Multiple TissueBlots I, III and IV from Clontech (Palo Alto, Calif.). A probe wasgenerated from a gel purified PCR product made from ZC21621 and ZC21622(SEQ ID NOS: 10 and 11, respectively) as primers and zsig81 as template,that had been radioactively labeled with REDIPRME™ DNA labeling kit(Amersham, Arlington Heights, Ill.) according to the manufacturer'ssuggestion. The probe was purified using a NUCTRAP push column(Stratagene). EXPRESSHYB™ (Clontech) solution was used forprehybridization and as a hybridizing solution for the Northern blots.Hybridization took place overnight at 65° C., and the blots were thenwashed in 2×SSC and 0.05% SDS at RT, followed by a wash in 0.1×SSC and0.1% SDS at 50° C. One major transcript was observed at size ofapproximately 5.0 kb and 1.7 kb. The larger message was determined tocontain approximately a 2.5 kb of 3′ untranslated region. Signals werepresent in Heart and liver, with decreased expression in lung, kidney,stomach, thyroid, spinal cord, trachea, adrenal, uterus, small intestineand colon tissues. The expression of zsig81 was also examined with HumanRNA Master blot (Clontech) as described above. Within heart tissue,zsig81 was localized to smooth muscle aorta using a ClontechCardiovascular MTN Blot, and mRNA isolated from primary cultured cells.

Example 2 Mapping Human zsig81 Chromosomal Location

[0172] zsig81 was mapped to human chromosome 7 using the commerciallyavailable “GeneBridge 4 Radiation Hybrid Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panelcontained DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). Mapping was relative to theWhitehead Institute/MIT Center for Genome Research's radiation hybridmap of the human genome (the “WICGR” radiation hybrid map) which wasconstructed with the GeneBridge 4 Radiation Hybrid Panel.

[0173] For the mapping of zsig81 with the “GeneBridge 4 RH Panel”, 20 μlreactions were set up in a 96-well microtiter plate (Stratagene, LaJolla, Calif.), and used in a “RoboCycler Gradient 96” thermal cycler(Stratagene). Each of the 95 PCR reactions consisted of 2 μl 10×KlenTaqPCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.),1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 μlsense primer, ZC22801 (SEQ ID NO: 12), 1 μl antisense primer, ZC22802(SEQ ID NO: 13), 2 μl “RediLoad” (Research Genetics, Inc., Huntsville,Ala.), 0.4 μl 50×Advantage KlenTaq Polymerase Mix (ClontechLaboratories, Inc.), 25 ng of DNA from an individual hybrid clone orcontrol and ddH₂O for a total volume of 20 μl. The reactions wereoverlaid with an equal amount of mineral oil and sealed. The PCR cyclerconditions were as follows: an initial 1 cycle 5 minute denaturation at95° C., 35 cycles of a 1 minute denaturation at 95° C., 1 minuteannealing at 56° C. and 1.5 minute extension at 72° C., followed by afinal I cycle extension of 7 minutes at 72° C. The reactions wereseparated by electrophoresis on a 2% agarose gel (GIBCO-BRL LifeTechnologies, Gaithersburg, Md.).

[0174] The results showed that zsig81 maps 8.12 cR_(—)3000 from theframework marker D7S 12 on the WICGR chromosome 7 WICGR radiation hybridmap. Proximal and distal framework markers were D7S512 and WI-5478respectively. The use of surrounding markers positions zsig81 in the7q32-q33 region on the integrated LDB chromosome 7 map (The GeneticLocation Database, University of Southhampton.

Example 3 Tissue Distribution for Mouse zsig81

[0175] A probe was constructed from full length polynucleotide sequenceof mouse zsig81, and was used to identify mRNA tissue distribution on aMouse Multiple Tissue Northern, a Mouse Dot Blot and a Mouse EmbryonicNorthern (Clontech). The probe was radioactively labeled with REDIPRIME™DNA labeling kit (Amersham, Arlington Heights, Ill.) according to themanufacturer's suggestion. The probe was purified using a NUCTRAP pushcolumn (Stratagene). EXPRESSHYB™ (Clontech) solution was used forprehybridization and as a hybridizing solution for the Northern blots.Hybridization took place overnight at 65° C., and the blots were thenwashed in 2×SSC and 0.05% SDS at RT, followed by a wash in 0.1×SSC and0.1% SDS at 65° C. One major transcript was observed at size ofapproximately 1.7 kb. Signals were present in liver, brain, heart andlung in adult tissue. In the embryonic tissue mRNA expression washighest on day 15, followed by decreased expression on day 17.

Example 4 Chromosome Mapping of Mouse zsig81

[0176] Murine zsig81 was mapped in mouse using the commerciallyavailable mouse T31 whole genome radiation hybrid (WGRH) panel (ResearchGenetics, Inc., Huntsville, Ala.) and Map Manager QT linkage analysisprogram. At P=0.0001, murine Zsig81 linked to the marker D12Mit201 witha LOD score of 13.3. D12Mit201 has been mapped at 23 cM on mousechromosome 12. This is at the lower border of a known region of syntenyor linkage conservation with the region of human chromosome 7 where thehuman form of zsig81 has been mapped.

[0177] The T31 WGRH panel contains DNAs from each of 100 radiationhybrid clones, plus two control DNAs (the 129aa donor and the A23recipient). For the mapping of murine zsig81 with the T31 WGRH panel, 20μl reactions were set up in 96-well microtiter plates (Stratagene, LaJolla, Calif.) and used in “RoboCycler Gradient 96” thermal cyclers(Stratagene). Each of the 102 PCR reactions consisted of 2 μl 10×KlenTaqPCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.),1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 μLsense primer, ZC21229 (SEQ ID NO: 18), 1 μl antisense primer, ZC21711(SEQ ID NO: 19), 2 μl “RediLoad” (Research Genetics, Inc., Huntsville,Ala.), 0.4 μl 50×Advantage KlenTaq Polymerase Mix (ClontechLaboratories, Inc.), 25 ng of DNA from an individual hybrid clone orcontrol and ddH20 for a total volume of 20 μl. The reactions wereoverlaid with an equal amount of mineral oil and sealed. The PCR cyclerconditions were as follows: an initial 1 cycle 5 minute denaturation at94° C., 35 cycles of a 45 seconds denaturation at 94° C., 45 secondsannealing at 62° C. and 1 minute and 15 seconds extension at 72° C. ,followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 2% agarose gel (LifeTechnologies, Gaithersburg, Md.).

Example 5 Expression Constructs for zsig81

[0178] A. Mammalian Expression Constructs

[0179] An expression plasmid containing all or part of a polynucleotideencoding zsig81 is constructed via homologous recombination. A fragmentof zsig81 cDNA is isolated using PCR that includes the polynucleotidesequence from nucleotide 1 to nucleotide 472 of SEQ ID NO: 1 withflanking regions at the 5′ and 3′ ends corresponding to the vectorssequences flanking the zsig81 insertion point. The primers for PCR eachinclude from 5′ to 3′ end: 40 bp of flanking sequence from the vectorand 17 bp corresponding to the amino and carboxyl termini from the openreading frame of zsig81.

[0180] Ten μl of the 100 μl PCR reaction is run on a 0.8% LMP agarosegel (Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μlof PCR reaction is precipitated with the addition of 5 μl 1 M NaCl and250 μl of absolute ethanol. The plasmid pCZR199 which has been cut withSmal. Plasmid pCZR199 was constructed from pZP9 (deposited at theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209, and is designated No. 98668) with the yeast geneticelements taken from pRS316 (deposited at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, and isdesignated No. 77145) pCZR199 is a mammalian expression vectorcontaining an expression cassette having the mouse metallothionein-1promoter, multiple restriction sites for insertion of coding sequences,a stop codon and a human growth hormone terminator. The plasmid also hasan E. coli origin of replication, a mammalian selectable markerexpression unit having an SV40 promoter, enhancer and origin ofreplication, a DHFR gene, the SV40 terminator, as well as the URA3 andCEN-ARS sequences required for selection and replication in S.cerevisiae.

[0181] One hundred microliters of competent yeast cells (S. cerevisiae)are independently combined with 10 μl of the various DNA mixtures fromabove and transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures are electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two300 μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura+ yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA precipitated with 600 μl ethanol (EtOH), followed by centrifugationfor 10 minutes at 4° C. The DNA pellet is resuspended in 10 μl H₂O.

[0182] Transformation of electrocompetent E. coli cells (DH10B,GibcoBRL) 35 is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10Bcells. The cells are electropulsed at 1.7 kV, 25 μF and 400 ohms.Following electroporation, 1 ml SOC (2% Bacto′ Tryptone (Difco, Detroit,Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2,10 mM MgSO4, 20 mM glucose) is plated in 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto Agar (Difco), 100 mg/LAmpicillin).

[0183] Individual clones harboring the correct expression construct forzsig81 are identified by restriction digest to verify the presence ofthe zsig81 insert and to confirm that the various DNA sequences havebeen joined correctly to one another. The insert of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing the Qiagen Maxi kit (Qiagen) according to manufacturer'sinstruction.

[0184] B. Baculovirus Expression Construct

[0185] Construction of pzBV/zSig81.CF (Baculovirus expression vector)

[0186] An expression vector, pZBVhzSig81, was prepared to express HumanzSig81 polypeptide in insect cells. A 483 bp fragment containingsequence for Human zSig81 and the coding sequence for a c-terminal FLAGtag (SEQ ID NO: 20) and encoded BspEI and XbaI restriction sites on the5′ and 3′ ends respectively, was generated by PCR amplification from aplasmid containing human zSig81 cDNA (pcDNA3d2 hzsig81-CF) using primerszc26461 (SEQ ID NO: 21) and zc26475 (SEQ ID NO: 22). The PCR reactionconditions were as follows: 25 cycles of 94° C. for 1 minute, 58° C. for1 minute, and 72° C. for 2 minutes; 1 cycle at 72° C. for 10 minutes;followed by a 4° C. soak. The digested vector was visualized by gelelectrophoreses, 0.8% agarose (EM Science, Gibbstown, N.J.) and purifiedusing the Qiaquick Gel Extraction Kit (Qiagen, Valencia, Calif.). About20 ng of the purified Human zSig81 fragment was ligated, at roomtemperature, overnight, to about 5 ng of purified pzBV3L vector. Onemicroliter of the ligation mixture was transformed into ElectrocompetantDH10B cells (Life Technologies, Gaithersburg, Md.). Eight clones werepicked and grown overnight in LB/Amp broth. Plasmids from the cloneswere purified by Qiaprep (Qiagen, Valencia, Calif.) mini prep kit. PCRwas used to analyse clones for insert using 5′ primer ZC2359 (SEQ ID NO:23) and 3′ primer ZC12581 (SEQ ID NO: 24). Each mini prep was diluted1:100 in distilled, sterile water and 1 μl used for a 50 μl reaction(reagents from Life Technologies, Gaithersburg, Md.). Twenty microlitersof each reaction was vizualized by gel electrophoresis, 1% agarose.Clone #1 was chosen to transform into cells DH10Bac to produce a“Bacmid”.

[0187] Baculovirus Expression Construct of Murine zSig 81 cee

[0188] A 549 bp fragment containing sequence for Murine zSig81 andencoded BamH1 and Xba1 restriction sites on the 5′ and 3′ ends,respectively, was generated by PCR amplification from a plasmidcontaining Murine zSig81 cDNA (described above) using primers ZC23433(SEQ ID NO: 25) and ZC23432 (SEQ ID NO:26) utilizing the Expand HighFidelity PCR System (Boehringer Mannheim, Indianapolis, Ind.) as permanufacturers instructions. The PCR conditions were as follows: 1 cycleof 94° C. for 4 minutes, followed by 25 cycles of 94° C. for 45 seconds,50° C. for 45 seconds, and 72° C. for 2 minutes; 1 cycle at 72° C. for10 min; followed by a 10° C. soak. A small portion of the PCR productwas visualized by gel electrophoresis (1% NuSieve agarose). Theremainder of the fragment was precipitated and resuspended in 5 ul ofH₂O. The fragment was then digested in a 50 μl vol. with BamH1 and Xba1restriction enzymes at 37° C. for 3 hrs, then run on agarose gel asdescribed above. About 17.5 nanograms of the restriction digestedzSig81M insert and about 53.8 ng of the corresponding vector wereligated overnight at 16° C.

[0189] Construction of Expression Vector pZBV37L hzSig81

[0190] An expression vector, pZBV37L hzSig81, was prepared to expressHuman zSig81 polypeptide in insect cells. A 483 bp fragment containingsequence for Human zSig81 and the coding sequence for a c-terminalglu-glu tag and encoded BspEI and XbaI restriction sites on the 5′ and3′ ends respectively, was generated by PCR amplification from a plasmidcontaining human zSig81 cDNA (pcDNA3d2 hzsig81-CF) using primers ZC26461(SEQ ID NO: 21) and ZC26475 (SEQ ID NO: 22). The PCR reaction conditionswere as follows: 25 cycles of 94° C. for 1 minute, 58° C. for 1 minute,and 72° C. for 2 minutes; 1 cycle at 72° C. for 10 minutes; followed bya 4° C. soak. The fragment was visualized by 1% gel electrophoresis. Theband was excised and purified using a QIAquick gel extraction kit(Qiagen) and ligated into a BspEI/Xbal digested baculovirus expressionvector, pZBV37L. The pZBV37L vector is a modification of the pFastBac1™(Life Technologies) expression vector, where the polyhedron promoter hasbeen removed and replaced with the late activating Basic ProteinPromoter and the EGT leader. The hzSig81 restriction digested fragmentand the pZBV37L vector were ligated overnight at room temperature in a4:1 ratio. Clones were prepared as described above.

[0191] Bacmid Production

[0192] Five mircroliters of the expression vectors described above weretransformed into 50 μl DH10Bac Max Efficiency competent cells(GIBCO-BRL, Gaithersburg, Md.) according to manufacturer'sspecifications. A color selection was used to identify those cellshaving Human zsig81 encoding donor insert that had incorporated into theplasmid (referred to as a “bacmid”). Those colonies, which were white incolor, were picked for analysis. Bacmid DNA was isolated from positivecolonies using the QiaVac Miniprep8 system (Qiagen) according themanufacturer's directions. Clones were screened for the correct insertby amplifying DNA using primers to the transposable element in thebacmid via PCR using primers ZC447 (SEQ ID NO:27) and ZC976 (SEQ IDNO:28). The PCR reaction conditions were as follows: 35 cycles of 94° C.for 45 seconds, 50° C. for 45 seconds, and 72° C. for 5 minutes; 1 cycleat 72° C. for 10 min.; followed by 4° C. soak. The PCR product was runon a 1% agarose gel to check the insert size. Those having the correctinsert were used to transfect Spodoptera frugiperda (Sf9) cells.

[0193] 1 μl of a positive clone was transformed into 20 μl DH10Bac MaxEfficiency competent cells (GIBCO-BRL, Gaithersburg, Md.) according tothe manufacturer's instruction by heat shock for 45 seconds in a 42° C.waterbath. The transformed cells were then diluted in 980 μl SOC media(2% Bacto™ Tryptone, 0.5% Bacto™ Yeast Extract, 10 ml 1M NaCl, 1.5 mMKCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) out grown in a shakingincubator at 37° C. for four hours and plated onto Luria Agar platescontaining 50 μg/ml kanamycin, 7 μg/ml gentamicin (Life Technologies),10 μg/ml tetracycline, IPTG (Pharmacia Biotech) and Bluo-Gal (LifeTechnologies). The plated cells were incubated for 48 hours at 37° C. Acolor selection was used to identify those cells having human zSig81encoding donor insert that had incorporated into the plasmid (referredto as a “bacmid”). Those colonies, which were white in color, werepicked for analysis by PCR using primers to the transposable element inthe bacmid with primers ZC447 (SEQ ID NO: 27) and ZC976 (SEQ ID NO: 28).The PCR reaction conditions were as follows: 35 cycles of 94° C. for 45seconds, 50° C. for 45 seconds, and 72° C. for 5 minutes; 1 cycle at 72°C. for 10 minutes; followed by a 4° C. soak. The PCR product was run ona 1% agarose gel to check for insert size. Those clones having thecorrect insert size were used to transfect Spodoptera frugiperda (Sf9)cells.

[0194] C. Expression of Human zsig81 in E. coli

[0195] Construction of zsig81 -MBP Fusion Expression VectorPTAP98/zsig81

[0196] An expression plasmid containing a polynucleotide encoding partof the human zsig81 fused N-terminally to maltose binding protein (MBP)was constructed via homologous recombination. A fragment of human zsig81cDNA (SEQ ID NO: 29) was isolated using PCR. Two primers were used inthe production of the human zsig81 fragment in a PCR reaction: (1)Primer ZC22990 (SEQ ID NO: 30), containing 40 bp of the vector flankingsequence and 24 bp corresponding to the amino terminus of the humanzsig81, and (2) primer ZC22991 (SEQ ID NO: 31), containing 40 bp of the3′ end corresponding to the flanking vector sequence and 21 bpcorresponding to the carboxyl terminus of the human zsig81. The PCRreaction conditions were as follows: 25 cycles of 94° C. for 30 seconds,55° C. for 30 seconds, and 72° C. for 2 minutes; followed by 4° C. soak,run in duplicate. Two μl of the 100 μl PCR reaction were run on a 1.0%agarose gel with 1×TBE buffer for analysis, and the expected band ofapproximately 500 bp fragment was seen. The remaining 90 μl of PCRreaction was combined with the second PCR tube precipitated with 400 μlof absolute ethanol to be used for recombining into the Smal cutrecipient vector pTAP98 to produce the construct encoding the MBP-zsig81fusion, as described below.

[0197] Plasmid pTAP98 was derived from the plasmids pRS316 and pMAL-c2.The plasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (HieterP. and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E.coli expression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rmB terminator. The vector pTAP98 was constructed usingyeast homologous recombination. 100 ng of EcoR1 cut 20 pMAL-c2 wasrecombined with 1 μg Pvul cut pRS316, 1 μg linker, and 1 μg Sca1/EcoR1cut pRS316. The linker consisted of 100 pmole of ZC19372 (SEQ ID NO:32); 1 pmole of ZC19351 (SEQ ID NO: 33); 1 pmole of ZC19352 (SEQ ID NO:34); and 100 pmole of ZC19371 (SEQ ID NO: 35) combined in a PCRreaction. Conditions were as follows: 10 cycles of 94° C. for 30seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds; followed by4° C. soak. PCR products were concentrated via 100% ethanolprecipitation.

[0198] One hundred microliters of competent yeast cells (S. cerevisiae)were combined with 10 μl of a mixture containing approximately 1 μg ofthe human zsig81 insert, and 100 ng of SmaI digested pTAP98 vector, andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixturewas electropulsed at 0.75 kV (5 kV/cm), infinite ohms, 25 μF. To eachcuvette was added 600 μl of 1.2 M sorbitol.

[0199] The yeast was then plated in two 300 μl aliquots onto two −URA Dplates and incubated at 30° C.

[0200] After about 48 hours, the Ura+ yeast transformants from a singleplate were resuspended in 1 ml H₂O and spun briefly to pellet the yeastcells. The cell pellet was resuspended in 1 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Fivehundred microliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C.. The DNA pellet was resuspendedin 100 μl H₂O.

[0201] Transformation of electrocompetent E. coli cells (MC1061,Casadaban et. al. J. Mol. Biol. 138, 179-207) was done with 1 μl yeastDNA prep and 40 μl of MC1061 cells. The cells were electropulsed at 2.0kV, 25 μF and 400 ohms. Following electroporation, 0.6 ml SOC (2% BactoiTryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was plated inone aliquot on LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

[0202] Individual clones harboring the correct expression construct forhuman zsig81 were identified by PCR screening using oligonucleotidesZC22990 (SEQ ID NO: 30) and ZC22991 (SEQ ID NO: 31). Reaction conditionswere as follows: 25 cycles of 94° C. for 30 seconds; 50° C. for 30seconds; 72° C. for one minute; then followed by a 4° C. soak. Of thepositive clones producing a band around 500 bp as seen using agarose gelelectrophoresis, 4 were then further screened by expression. Cells weregrown in Superbroth II (Becton Dickinson) with 100 μg/ml of ampicillinovernight. 50 μl of the overnight culture was used to inoculate 2 ml offresh Superbroth II+100 μg/ml ampicillin. Cultures were grown at 37° C.,shaking for 2 hours. 1 ml of the culture was induced with 1 mM IPTG. 2-4hours later the 250 μl of each culture was mixed with 250 μl acid washedglass beads and 250 μl Thorner buffer with 5% BME and dye(8M urea, 100mM Tris pH7.0, 10% glycerol, 2mM EDTA, 5% SDS). Samples were vortexedfor one minute and heated to 65° C. for 10 minutes. 20 μl were loadedper lane on a 4%-12% PAGE gel (NOVEX). Gels were run in 1×MES buffer.The positive clones were subjected to sequence analysis. The correctclone was designated pTAP139.

Example 6 Expression of zsig81

[0203] A. Mammalian Expression of zsig81

[0204] CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,1986) are plated in 10 cm tissue culture dishes and allowed to grow toapproximately 50 to 70% confluency overnight at 37° C. , 5% CO₂, inHam's F12/FBS media (Ham's F12 medium, (Gibco BRL, Gaithersburg, Md.),5% fetal bovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRHBiosciences, Lenexa, Kans.), 1% sodium pyruvate (Gibco BRL)). The cellsare then transfected with the plasmid zsig81/pCZR199, usingLipofectamine™ (Gibco BRL), in serum free (SF) media formulation (Ham'sF12, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1%L-glutamine and 1% sodium pyruvate). zlipo3/pCZR199 is diluted into 15ml tubes to a total final volume of 640 μl with SF media. 35 μl ofLipofectamine™ (Gibco BRL) is mixed with 605 μl of SF medium. TheLipofectamine™ mix is added to the DNA mix and allowed to incubateapproximately 30 minutes at room temperature. Five milliliters of SFmedia is added to the DNA:Lipofectamine™ mixture. The cells are rinsedonce with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells are incubated at 37° C. for five hours, then6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate. Theplates are incubated at 37° C. overnight and the DNA:Lipofectamine™mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 2post-transfection, the cells are split into the selection media(nucleoside-free Alpha MEM/dialyzed FBS media with the addition of 50 nMmethotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates at1:10, 1:20 and 1:50. The cells are refed at day 5 post-transfection withfresh selection media. Approximately 10 days post-transfection, two 150mm culture dishes of methotrexate resistant colonies from eachtransfection are trypsinized and the cells are pooled and plated into aT-162 flask and transferred to large scale culture for scale-up anddilution cloning.

[0205] Cells are plated for subcloning at a density of 0.5, 1 and 5cells per well in 96 well dishes in selection medium and allowed to growout for approximately two weeks. The wells are checked for evaporationof medium and brought back to 200 μl per well as necessary during thisprocess. When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the are fed with fresh selection medium. The supernatantis applied to nitrocellulose filter in a dot blot apparatus and thefilter is treated at 100° C. in a vacuum oven to denature the protein.The filter was incubated in 625 mM tris glycine, pH 9.1, 5 mMβmercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fat dry milkWestern A Buffer (0.25% gelatin, 50 mM TrisHCl pH 7.4, 150 mM NaCl, 5 mMEDTA, 0.05% Igepal CA-630) overnight at 4° C. on a rotating shaker. Thefilter was incubated with the antibody-HRP conjugate in 2.5% non-fat drymilk Western A buffer for 1 hour at room temperature on a rotatingshaker. The filter was washed three times at room temperature in PBSplus 0.01% Tween 20, 15 minutes per wash. The filter was developed withECL reagent according to manufacturer's directions (Amersham, ArlingtonHeights, Ill.) and exposed to film (Hyperfilm ECL, (Amersham)approximately 5 minutes. Positive clones are trypsinized from the 96well dish and transferred to 6 well dishes in selection medium forscaleup and analysis by Western blot.

[0206] B. Yeast Expression

[0207] Expression of zsig81 in Pichia methanolica utilizes theexpression system described in commonly-assigned WIPO publication WO97/17450. An expression plasmid containing all or part of apolynucleotide encoding zsig81 is constructed via homologousrecombination.

[0208] An expression vector is built from pCZR190 to express N-terminaltagged zsig81 polypeptides. The pCZR190 vector contains the AUGIpromoter, followed by the aFpp leader sequence and an amino-terminalpeptide tag (FLAG; SEQ ID NO: 20), followed by a blunt-ended Sma Irestriction site, a translational STOP codon, followed by the AUG1terminator, the ADE2 selectable marker, and finally the AUG1 3′untranslated region. Also included in this vector are the URA3 andCEN-ARS sequences required for selection and replication in S.cerevisiae, and the AmpR and colE1 ori sequences required for selectionand replication in E. coli. For each construct two linkers are prepared,and along with zsig81, are homologously recombined into the yeastexpression vectors described herein.

[0209] One hundred microliters of competent yeast cells (S. cerevisiae)are independently combined with 10 μl of the various DNA mixtures fromabove and transferred to a 0.2 cm electroporation cuvette. The yeast/DNAmixtures are electropulsed at 0.75 kV (5 kV/cm), ∞ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol and the yeast is plated in two300 μl aliquots onto two URA-D plates and incubated at 30° C.

[0210] After about 48 hours, the Ura+ yeast transformants from a singleplate are resuspended in 1 ml H₂O and spun briefly to pellet the yeastcells. The cell pellet is resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenolchloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA precipitated with 600 μl ethanol (EtOH), followed by centrifugationfor 10 minutes at 4° C. The DNA pellet is resuspended in 100 μl H₂O.

[0211] Transformation of electrocompetent E. coli cells (DH10B,GibcoBRL) is done with 0.5-2 μl yeast DNA prep and 40 ul of DH10B cells.The cells is electropulsed at 2.0 kV, 25 mF and 400 ohms. Followingelectroporation, 1 ml SOC (2% Bacto′ Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCI, 10 mM MgCl2, 10 mMMgSO4, 20 mM glucose) is plated in 250 μl aliquots on four LB AMP plates(LB broth (Lennox), 1.8% Bacto′ Agar (Difco), 100 mg/L Ampicillin).

[0212] Individual clones harboring the correct expression construct forzsig81 are identified by PCR analysis or restriction digest to verifythe presence of the zsig81 insert and to confirm that the various DNAsequences have been joined correctly to one another. The insert ofpositive clones are subjected to sequence analysis. Larger scale plasmidDNA is isolated using the Qiagen Maxi kit (Qiagen) according tomanufacturer's instruction, and the DNA is digested with Not I toliberate the Pichia-zsig81 expression cassette from the vector backbone.The Not I-restriction digested DNA fragment is then transformed into thePichia methanolica expression host, PMAD16. This is done by mixing 100ml of prepared competent PMAD16 cells with 10 μg of Not I restrictiondigested zsig81 and transferred to a 0.2 cm electroporation cuvette. Theyeast/DNA mixture is electropulsed at 0.75 kV, 25 mF, infinite ohms. Tothe cuvette is added 1 ml of 1× Yeast Nitrogen Base and 500 ml aliquotsare plated onto two ADE DS (0.056% -Ade -Trp -Thr powder, 0.67% yeastnitrogen base without amino acids, 2% D-glucose, 0.5% 200× tryptophan,threonine solution, and 18.22% D-sorbitol) plates for selection andincubated at 30° C . Clones are picked and screened via Western blot forhigh-level zsig81 expression and fermented.

[0213] C. Baculovirus Expression of zsig81

[0214] Expression of pzBV/zSig81.CF (Baculovirus Expression Vector)

[0215] An expression vector, pZBVhzSig81, was prepared to express HumanzSig81 polypeptide in insect cells as described in Example 2B. Sf9 cellswere seeded at 1 million cells per 35 mm plate and allowed to attach for1 hour at 27° C.. Five microliters of bacmid DNA was diluted with 100 μlSf-900 II SFM (Life Technologies). 25 μl of CellFECTIN™ Reagent (LifeTechnologies) was diluted with 100 μl Sf-900 II SFM. The bacmid DNA andlipid solutions were gently mixed and incubated 30-45 minutes at roomtemperature. The media from one plate of cells were aspirated, the cellswere washed 1×X with 2 ml fresh Sf-900 II SFM media. Eight hundredmicroliters of Sf-900 II SFM was added to the lipid-DNA mixture. Thewash media was aspirated and the DNA-lipid mix added to the cells. Thecells were incubated at 27° C. for 24 hours. The DNA-lipid mix wasaspirated and 2 ml of Sf-900 II media was added to each plate. Theplates were incubated at 27° C., 90% humidity, for 5 days, after whichthe virus was harvested.

[0216] Sf9 cells were seeded as above and 200 μl of post transfectionsupernatant was added and cultures were allowed to proceed for 72 hrsafter which time the virus was harvested.

[0217] Sf9 cells were seeded as above and 20 μl of the Primary viralstock was added. Cultures were incubated at 27° C. for 9 days, afterwhich time the virus was harvested according to standard methods knownin the art.

[0218] 20 μl of Secondary Amplified virus stock was placed on SF9s at500,000 cells per well in 50 mls of SF900II media in a 250 ml vol shakeflask for 96 hrs, and virus was harvested as above.

[0219] Presence of predicted molecular weight protein in the cell lysatewas determined by western analysis using anti-FLAG primary monoclonalantibody and goat anti-mouse HRP secondary. Material was alsoimmunoprecipatated using anti-EE antibody conjugated to sepharose andsubmitted for n-terminal signal peptidase cleavage point analysis.

[0220] Baculovirus Expression of Murine zSig 81 cee

[0221] An expression vector, was prepared to express Murine zSig81polypeptides in insect cells as described in Example 5. A 50 ml cultureof Trichoplusia ni (HiS) cells at 2.2×10⁶ cells/ml was infected with 22ml of primary amplification supernatant. The infection culture wasallowed to progress for 48 hrs and the cells pelleted via centrifugationand frozen at −20° C . A hypotonic lysis of the pellet was performed asfollows: pellet thawed, 2.5 ml of lysis buffer (0.02M Tris-HCl ph8.3,0.001 M EDTA, ,00DTT, 1 mM Pefabloc, 500 nM Aprotonin, 4 μM Leupeptin, 4μM E-64, 1% NP-40) was used to resuspend the cells. Lysis was allowed toprogress at 4° C. for 15 min. The cellular debris was spun out, and thesupernatant was collected and incubated with sepharose beadsw/conjugated anti-EE antibody. The beads were washed and submitted tofor signal peptidase cleavage point determination.

[0222] Sample Preparation: Murine zsig81CEE samples were supplied onanti-EE beads. The beads were placed in reducing SDS PAGE sample bufferand on a boiling water bath before running on SDS PAGE. (Novex SDS PAGEsystem and supplies, 4-12% bis-tris MES NuPAGE). These wereelectrotransferred to Novex PVDF membrane, and then coomassie bluestained. Corresponding anti-EE westerns were performed to identify whichbands to excise for N-terminal protein sequencing.

[0223] Human zsig81CF samples were supplied on anti-FLAG beads. Thebeads were placed in both reducing and non-reducing SDS PAGE samplebuffer and on a boiling water bath before running on SDS PAGE. (NovexSDS PAGE system and supplies, 4-12% bis-tris MES NuPAGE). These wereelectrotransferred to Novex PVDF membrane, and then coomassie bluestained. Corresponding anti-FLAG westerns were performed to identifywhich bands to excise for N-terminal protein sequencing.

[0224] N-terminal sequence analyses were performed on a Models 476A and494 Protein Sequencer Systems from Perkin Elmer Applied BiosystemsDivision, Foster City, Calif. Data analysis was performed with AppliedBiosystems Model 610A Data Analysis System for Protein Sequencing,version 2.1a (Applied Biosystems Inc. Foster City, Calif.). Mostsupplies and reagents used were from Applied Biosystems Inc.

[0225] The predicted mature start is at Serl8 of the human precursorform and at Thr18 of the murine precursor form.

[0226] For the murine form from BV with the C-terminal EE tag (SEQ IDNO: 36) one experimental mature start is at Ser20 of the precursor form.There is an indication that another experimental mature start at Arg22is present. This second call is ambiguous due to contaminating histonesample.

[0227] Human zsig81CF from BV resulted in an experimental mature startat Ser18 of the precursor sequence which is also the predicted maturestart. Within this sample there were also minor amounts of precursorsequence starting at His21 and Met1.

[0228] Expression of Arg22 Human zSig81

[0229] Based on these results and the observation that homology of humanand murine zSig81 are divergent from the initiating methinonine and thenconverge at arginine 22 led us to prepare baculovirus expression vectorsfusing the ecdysteroid UDP-glucasyltransferase (EGT) leader sequence toarginine 22 of both human and murine zSig81.

[0230] Sequence analysis of the human zSig81. CEE revealed that all ofthe zSig81 protein was processed as predicted, leaving Arg 22 as theamino terminal 5 amino acid of the mature peptide. PAGE and westernanalysis indicate mature zSig81.CEE as isolated, is approximately 50%disulfide linked dimer and 50% monomer.

[0231] Bacterial Expression of Human zsig81.

[0232] One microliter of pTAP139 sequencing DNA was used to transformstrain W3110 (ATCC No. 27325). The cells were electropulsed at 2.0 kV,25 μF and 400 ohms. Following electroporation, 0.6 ml SOC (2% BactoëTryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mMNaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was plated inone aliquot on LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

[0233] Individual colonies were expressed. Cells were grown inSuperbroth II (Becton Dickinson) with 100 μg/ml of ampicillin overnight.50 μl of the overnight culture was used to inoculate 2 ml of freshSuperbroth II+100 μg/ml ampicillin. Cultures were grown at 37° C.,shaking for 2 hours. 1 ml of the culture was induced with 1 mM IPTG. 2-4hours later the 250 μl of each culture was mixed with 250 μl acid washedglass beads and 250 μl Thorner buffer with 5% BME and dye (8M urea, 100mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples were vortexedfor one minute and heated to 65° C. for 10 minutes. 20 μl were loadedper lane on a 4%-12% PAGE gel (NOVEX). Gels were run in 1×MES buffer.

Example 7

[0234] Protein Purification of zsig81

[0235] Unless otherwise noted, all operations will be carried out at 4°C. A total of 25 liters of conditioned medium from chinese hamster ovary(CHO) cells or baby hamster kidney cells (BHK) is be sequentiallysterile filtered through a 4 inch, 0.2 mM Millipore (Bedford, Mass.)OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, Mich.) Supercap50. The material is then be concentrated to about 1.3 liters using aMillipore ProFlux A30 tangential flow concentrator fitted with a 3000kDa cutoff Amicon (Bedford, Mass.) S10Y3 membrane. The concentratedmaterial is sterile-filtered with the Gelman filter again as describedabove. A mixture of protease inhibitors is added to the concentratedconditioned medium to final concentrations of 2.5 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis,Mo.), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, Ind.),0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc(Boehringer-Mannheim).

[0236] Generic screening for protein capture is carried out using aBioCad 700E, Sprint, or Vision workstation (PE Biosystems, Framingham,Mass.) using the column screening module according to the manufacturer'sinstructions. A 50 ml sample of the concentrated CHO conditioned mediumis brought to the appropriate pH by in-line dilution with screeningbuffer (25 mM Tris, 25 mM MOPS, 25 mM MES, and 25 mM acetate adjusted tothe appropriate pH as described below) and pumped sequentially at a flowrate of 2-5 ml/min onto a 1.7 ml Poros HS (PE Biosystems) columnequilibrated at pH 4.0, 5.0, and 6.0, onto a 1.7 ml Poros HQ (PEBiosystems) column equilibrated at pH 7.0, 8.0, and 9.0, onto a 1.7 mlPoros HE (PE Biosystems) column at pH 7.4, and onto a 1.7 ml Poros HP2(PE Biosystems) column equilibrated at pH 7.4 and 1.0-4.0 M NaCl. Aftersample application, each column is washed with the appropriateequilibration buffer and when the absorbance at 280 nm of the effluentis below 0.05, the Poros HS, HQ, and HE columns are eluted stepwise with1.0-2.0 M NaCl. The Poros HP2 column is eluted stepwise with water. 1.0ml fractions is collected and the target protein in each of the columneluates is identified by the automated proteolysis-mass spec proceduredescribed below. Positive identities are confirmed by SDS-PAGE analysisof each eluate fraction according to standard procedures.

[0237] Once the binding conditions are established for a particularprotein, these conditions are used for its large batch purification.Purity at each step of the purification is assessed by SDS-PAGE andWestern blotting with anti-zsig81 antibodies directed against theMBP-fusion of the target protein.

[0238] Proteins eluted as described above are detected independent ofwestern blotting or other antibody related strategies. The presence ofthe desired protein is determined as either a single component or in acomplex mixture by analysis of the eluate of a column, collected overseveral fractions and resulting in a relative quantitation of the amountof zsig81 protein present in each fraction.

[0239] The system uses a stepwise combination of proteolytic digestionof protein samples (module 1), chromatographic separation (module 2) andmass spectral analysis (module 3) of the digestion mixture. The threemodules of this process are used individually for analysis of proteinsamples in a manual fashion, resulting in maximal data output, or in thestepwise process of 3 modules in a fully automated set- up, resulting inmaximal high throughput.

[0240] In the automated set-up, module 1 and 2 are combined in theINTEGRAL Workstation (PE Biosystems, Farmington, Mass.) which is on-lineconnected to module 3. Module 3 is an LCQ ion-trap mass spectrometer(Finnigan, San Jose, Calif.) equipped with an electrospray source.

[0241] To maximize data output, module 1 and 2 are separated and theproteolytic digestion of samples is removed from the automatedprocedure. A MAGIC HPLC system (Michrom BioResources, Inc., Auburn,Calif.) serves as module 2. Samples are injected either manually or viaautoinjector. Module 3 is an LCQ ion-trap mass spectrometer equippedwith an electrospray source. Module 3 is on-line connected to module 2.

[0242] Module 1: Proteolytic Digestion

[0243] Typically, samples are proteolytically digested with trypsin,however, other proteases with defined specificity can be utilized. Ifnecessary, samples are filtered or centrifuged to remove aggregates orother potential particulate matter. In some cases, samples are appliedto a size exclusion step by filtration prior to analysis to simplify theresulting digestion mixture and make the identification of peptidesrelated to the desired protein easier. All necessary buffer adjustmentsare made before proteolytic digestion.

[0244] In the automated set-up, the samples are digested on-line on animmobilized trypsin column (PE Biosystems). The injection onto thecolumn is done using the INTEGRAL autoinjector and the resultingpeptides are chromatographically separated on module 2.

[0245] In the manual approach, samples are digested overnight insolution and injected by hand or via autoinjector onto module 2.

[0246] Module 2: Chromatographic Separation

[0247] The chromatographic separation of peptides is carried out on a 1mm ID reverse phase (POROS, PE Biosystems) column (LC-Packings, SanFrancisco, Calif.). Typically, the column is eluted with atrifluoroacetic acid (TFA)/water, TFA/acetonitrile gradient and theelution of peptides is monitored by UV. In the automated, as well as themanual approach, peptides are analyzed on-line on module 3 as they eluteoff the column.

[0248] Module 3: Mass Spectral Analysis

[0249] The mass spectral analysis of peptides is carried out using the“triple play” approach. First, full mass range scans are taken as thecolumn eluate is sprayed into the source of the mass spectrometer. If asignal above a predetermined intensity threshold is detected, theinstrument switches to a setting which provides a high resolution massmeasurement, followed by an MS/MS scan.

[0250] The MS/MS scan provides the fragmentation pattern which is usedto derive the primary sequence of the peptide. Peptide sequences arethen used for the identification of the protein. Typically, primarysequence and the nature of the protein is determined using the searchalgorithm SEQUEST (Finnigan). Mass spectral sample and data analysis arecarried out automatically. If necessary, data interpretation to derivepeptide sequences is done manually and the protein is identified using avariety of standard database search algorithms.

[0251] Ion intensities and number of peptides detected for one proteinare used to determine the relative abundance of this protein indifferent fractions.

[0252] If the mass spectral analysis is carried out on all ions observedleading to the analysis of all components in the digestion mixture. Inorder to simplify the analysis, the mass spectrometer is typically setto analyze only those ions which can be expected following theproteolysis of the desired protein. Through this filter, the analysisbecomes amenable to very complex mixtures which potentially contains thedesired protein as only a minor component.

[0253] Purification from insect cells Sf9

[0254] Sf9 cells infected with the expression virus, pzBV37Lhzsig81,from 25 one liter of culture, expressing hzSig81.CEE were lysed byincubation in 150 mM NaCl, 50 mM Tris (pH 8.0), 1% NP-40, COMPLETE™protease inhibitor cocktail (Roche, Indianapolis, Ind.) for one hour at4° C. The lysate was cleared of cellular debris by spinning in acentrifuge at 550×g for 15 minutes at 4° C. The supernatent was combinedwith 4 mL anti-EE monoclonal antibody/sepharose resin and incubatedovernight at 4° C. The antibody-resin was collected, washed with 20column volumes of ice cold PBS, and human zsig81.CEE was eluted with 1mL 100 mM glycine pH 2.0. The glycine eluate was neutralized with 100 mLTris pH 8.8 and then dialyzed against 8L of PBS. The dialyzed eluatecontained 500 mg/mL protein of which, by PAGE and western analysis,approximately 70% was human zsig81.CEE (zsig81 concentrationapproximately 350 mg/mL).

[0255] Sequence analysis of the human zSig81.CEE revealed that all ofthe zSig81 protein was processed as predicted, leaving Arg 22 as theamino terminal amino acid of the mature peptide. PAGE and westernanalysis indicate mature zSig81.CEE as isolated, is approximately 50%disulfide linked dimer and 50% monomer.

Example 8

[0256] Adenoviral Expression of zsig81

[0257] The protein coding region of zsig81 is amplified by PCR usingprimers that added FseI and AscI restriction sties at the 5′ and 3′termini respectively. PCR primers are used with a template containingthe full-length zsig81 cDNA in a PCR reaction as follows: one cycle at95° C. for 5 minutes; followed by 15 cycles at 95° C. for 1 min., 58° C.for 1 min., and 72° C. for 1.5 min.; followed by 72° C. for 7 min.;followed by a 4° C. soak. The PCR reaction product is loaded onto a 1.2% (low melt) SeaPlaque GTG (FMC, Rockland, Me.) gel in TAE buffer. Thezsig81 PCR product is excised from the gel and purified using theQIAquick™ PCR Purification Kit gel cleanup kit as per kit instructions(Qiagen). The PCR product is then digested, phenol/chloroform extracted,EtOH precipitated, and rehydrated in 20 ml TE (Tris/EDTA pH 8). Thezsig81 fragment is then ligated into the cloning sites of the transgenicvector pTG12-8 (See, description herein) and transformed into DH10Bcompetent cells by electroporation. Clones containing zsig81 areidentified by plasmid DNA miniprep followed by digestion. A positiveclone is confirmed by direct sequencing.

[0258] The zsig81 cDNA is released from a TG12-8 vector using FseI andAscI enzymes. The cDNA is isolated on a 1% low melt SeaPlaque GTG™ (FMC,Rockland, Me.) gel, and is then excised from the gel. The gel slice ismelted at 70° C., extracted twice with an equal volume of Tris bufferedphenol, and EtOH precipitated. The DNA is resuspended in 10 μl H₂O.

[0259] The zsig81 cDNA is cloned into the FseI-AscI sites of a modifiedpAdTrack CMV (He et al., PNAS 95:2509-2514, 1998). This constructcontains the GFP marker gene. The CMV promoter driving GFP expressionwas replace with the SV40 promoter and the SV40 polyadenylation signalhas been replaced with the human growth hormone polyadenylation signal.In addition, the native polylinker was replaced with FseI, EcoRV, andAscI sites. This modified form of pAdTrach CMV was named pZyTrack.Ligation is performed using the Fast-Link™ DNA ligation and screeningkit (Epicentre Technologies, Madison, Wis.). In order to linearize theplasmid, approximately 5 μg of the pZyTrack zsig81 plasmid is digestedwith PmeI. Approximately 1 μg of the linearized plasmid is cotransformedwith 200 ng of supercoiled pAdEasy (He et al., supra.) into BJ5183cells. The co-transformation is done using a Bio-Rad Gene Pulser at 2.5kV, 200 ohms and 25mFa. The entire co-transformation is plated on 4 LBplates containing 25 μg/ml kanamycin. The smallest colonies are pickedand expanded in LB/kanamycin and recombinant adenovirus DNA identifiedby standard DNA miniprep procedures. Digestion of the recombinantadenovirus DNA with FseI-AscI confirms the presence of zsig81. Therecombinant adenovirus miniprep DNA is transformed into DH10B competentcells and DNA prepared using a Qiagen maxi prep kit as per kitinstructions.

[0260] Approximately 5 μg of recombinant adenoviral DNA is digested withPacI enzyme (New England Biolabs) for 3 hours at 37° C. in a reactionvolume of 100 μl containing 20-30U of PacI. The digested DNA isextracted twice with an equal volume of phenol/chloroform andprecipitated with ethanol. The DNA pellet is resuspended in 10 μldistilled water. A T25 flask of QBI-293A cells (Quantum Biotechnologies,Inc. Montreal, Qc. Canada), inoculated the day before and grown to60-70% confluence, are transfected with the PacI digested DNA. ThePacI-digested DNA is diluted up to a total volume of 50 μl with sterileHBS (150 mM NaCl, 20 mM HEPES). In a separate tube, 20 μl DOTAP(Boehringer Mannheim, 1 mg/ml) is diluted to a total volume of 100 μlwith HBS. The DNA is added to the DOTAP, mixed gently by pipeting up anddown, and left at room temperature for 15 minutes. The media is removedfrom the 293A cells and washed with 5 ml serum-free MEM-alpha (GibcoBRL) containing 1 mM Sodium Pyruvate (GibcoBRL), 0.1 mM MEMnon-essential amino acids (GibcoBRL) and 25 mM HEPES buffer (GibcoBRL).5 ml of serum-free MEM is added to the 293A cells and held at 37° C. TheDNA/lipid mixture is added drop-wise to the T25 flask of 293A cells,mixed gently and incubated at 37° C. for 4 hours. After 4 h the mediacontaining the DNA/lipid mixture is aspirated off and replaced with 5 mlcomplete MEM containing 5% fetal bovine serum. The transfected cells aremonitored for Green Fluorescent Protein (GFP) expression and formationof foci, i.e., viral plaques.

[0261] Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells expressing the GFP protein start to form foci.These foci are viral “plaques” and the crude viral lysate is collectedby using a cell scraper to collect all of the 293A cells. The lysate istransferred to a 50 ml conical tube. To release most of the virusparticles from the cells, three freeze/thaw cycles are done in a dryice/ethanol bath and a 37° C. waterbath.

[0262] The crude lysate is amplified (Primary (1°) amplification) toobtain a working “stock” of zsig81 rAdV lysate. Ten 10cm plates ofnearly confluent (80-90%) 293A cells are set up 20 hours previously, 200μl of crude rAdV lysate added to each 10 cm plate and monitored for 48to 72 hours looking for CPE under the white light microscope andexpression of GFP under the fluorescent microscope. When all of the 293Acells show CPE (Cytopathic Effect) this 1° stock lysate is collected andfreeze/thaw cycles performed as described under Crude rAdV Lysate.

[0263] Secondary (2°) Amplification of zsig81 rAdV is obtained asfollows: Twenty 15 cm tissue culture dishes of 293A cells are preparedso that the cells were 80-90% confluent. All but 20 mls of 5% MEM mediais removed and each dish is inoculated with 300-500 μl 1° amplified rAdvlysate. After 48 hours the 293A cells are lysed from virus productionand this lysate is collected into 250 ml polypropylene centrifugebottles and the rAdV purified.

[0264] NP-40 detergent is added to a final concentration of 0.5% to thebottles of crude lysate in order to lyse all cells. Bottles are placedon a rotating platform for 10 min. agitating as fast as possible. Thedebris is pelleted by centrifugation at 20,000×G for 15 minutes. Thesupernatant is transferred to 250 ml polycarbonate centrifuge bottlesand 0.5 volumes of 20% PEG8000/2.5M NaCl solution added. The bottles areshaken overnight on ice. The bottles are centrifuged at 20,000×G for 15minutes and supernatant discarded into a bleach solution. The whiteprecipitate forms in two vertical lines along the wall of the bottle oneither side of the spin mark and is precipitated virus/PEG. Using asterile cell scraper, the precipitate from 2 bottles is resuspended in2.5 ml PBS. The virus solution is placed in 2 ml microcentrifuge tubesand centrifuged at 14,000×G in the microfuge for 10 minutes to removeany additional cell debris. The supernatant from the 2 mlmicrocentrifuge tubes is transferred into a 15 ml polypropylene snapcaptube and adjusted to a density of 1.34 g/ml with cesium chloride (CsCl).The volume of the virus solution is estimated and 0.55 g/ml of CsCladded. The CsCl is dissolved and 1 ml of this solution weighed. Thesolution is transferred polycarbonate thick-walled centrifuge tubes 3.2ml (Beckman) and spun at 80,000 rpm (348,000×G) for 3-4 hours at 25° C.in a Beckman Optima TLX microultracentrifuge with the TLA-100.4 rotor.The virus forms a white band. Using wide-bore pipette tips, the virusband is collected.

[0265] The virus from the gradient will have a large amount of CsCl,which must be removed before it can be used on cells. Pharmacia PD-10columns prepacked with Sephadex G-25M (Pharmacia) are used to desalt thevirus preparation. The column is equilibrated with 20 ml of PBS. Thevirus is loaded and allowed to run into the column. Five ml of PBS isadded to the column and fractions of 8-10 drops collected. The opticaldensities of 1:50 dilutions of each fraction is determined at 260 nm ona spectrophotometer, and a clear absorbance peak identified. Thesefractions are pooled and the optical density (OD) of a 1:25 dilutiondetermined. A formula is used to convert OD into virus concentration:(OD at 260nm)(25)(1.1×10¹²)=virions/ml. The OD of a 1:25 dilution of thezsig81 rAdV was 0.221, giving a virus concentration of 6×10¹²virions/ml.

[0266] To store the virus, glycerol is added to the purified virus to afinal concentration of 15%, mixed gently and stored in aliquots at −80°C.

[0267] A protocol developed by Quantum Biotechnologies, Inc. (Montreal,Qc. Canada) is followed to measure recombinant virus infectivity.Briefly, two 96-well tissue culture plates are seeded with 1×104 293Acells per well in MEM containing 2% fetal bovine serum for eachrecombinant virus to be assayed. After 24 hours 10-fold dilutions ofeach virus from 1×10⁻² to 1×10⁻¹⁴ are made in MEM containing 2% fetalbovine serum. 100 μl of each dilution is placed in each of 20 wells.After 5 days at 37° C., wells are read either positive or negative forCytopathic Effect (CPE) and a value for “Plaque Forming Units/ml” (PFU)is calculated.

[0268] TCID₅₀ formulation used is as per Quantum Biotechnologies, Inc.,above. The titer (T) is determined from a plate where virus used isdiluted from 10⁻² to 10⁻¹⁴, and read 6 days after the infection. At eachdilution a ratio (R) of positive wells for CPE per the total number ofwells is determined.

[0269] To Calculate titer of the undiluted virus sample: the factor,“F”=1+d(S−0.5); where “S” is the sum of the ratios (R); and “d” is Log10of the dilution series, for example, “d” is equal to 1 for a ten-folddilution series. The titer of the undiluted sample isT=10^((1+F))=TCID₅₀/ml. To convert TCID₅₀/ml to pfu/ml, 0.7 issubtracted from the exponent in the calculation for titer (T). Thezsig81 adenovirus had a titer of 7.1×10¹⁰ pfu/ml.

Example 9 Transgenic Expression

[0270] Transgenic animals expressing zsig81 genes are made using adult,(,2-8 months, (CS7BL/6×C3H/N f1Taconic Farms)), prepubescent fertilefemales (donors) (C57BIJ6×C3H/N f1, 4-5 weeks, (Taconic Farms)) andadult fertile females (C57BL/6×C3H/N f1, 2-4 months, (Taconic Farms) asparents.

[0271] The donors are injected with approximately 8 IU/mouse of PregnantMare's Serum gonadotrophin (Sigma, St. Louis, Mo.) I.P., and 46-47 hourslater, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) areadministered I.P. to induce superovulation. Fertilized eggs arecollected and stored in a 37° C./5% CO₂ incubator until microinjection.

[0272] 10-20 micrograms of plasmid DNA containing a cDNA of the zsig81gene is linearized, gel-purified, and resuspended in 10 mM Tris pH 7.4,0.25 mM EDTA pH 8.0, at a final concentration of 5-10 nanograms permicroliter for microinjection. Plasmid DNA is microinjected intoharvested eggs and are penetrated with an injection needle, into one orboth of the haploid pronuclei.

[0273] The following day 2-cell embryos are transferred intopseudopregnant recipients. The recipients are returned to cages inpairs, and allowed 19-21 days gestation. After birth, 19-21 dayspostpartum is allowed before weaning. The 25 weanlings are sexed andplaced into separate sex cages, and a 0.5 cm biopsy (used forgenotyping) is snipped off the tail with clean scissors.

[0274] Genomic DNA is prepared from the tail snips using a Qiagen Dneasykit following the manufacturer's instructions. Genomic DNA is analyzedby PCR using primers designed to the human growth hormone (hGH) 3′ UTRportion of the transgenic vector. A region unique to the human sequenceis identified from an alignment of the human and mouse growth hormone 3′UTR DNA sequences, ensuring that the PCR reaction does not amplify themouse sequence. Primers ZC17251 (SEQ ID NO: 14) and ZC17252 (SEQ ID NO:15) amplify a 368 base pair fragment of hGH. In addition, primersZC17156 (SEQ ID NO: 16) and ZC17157 (SEQ ID NO: 17), which hybridize tovector sequences and amplify the cDNA insert, is often used along withthe hGH primers. In experiments, DNA from animals positive for thetransgene generate two bands, a 368 base pair band corresponding to thehGH 3′ UTR fragment and a band of variable size corresponding to thecDNA insert.

[0275] Once animals are confirmed to be transgenic (TG), they are bredback with C57B1/6 wild-type mates. As pups are born and weaned, thesexes are separated, and their tails snipped for genotyping.

[0276] To check for expression of a transgene in a live animal, a smallpartial hepatic biopsy is collected. The collected liver biopsy istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice.

[0277] Analysis of the mRNA expression level of each transgene is doneusing an RNA solution hybridization assay.

Example 10 Bone Marrow Assay of zsig81

[0278] A. Isolation of Non-Adherent Low Density Marrow Cells:

[0279] Fresh mouse femur aspirate (marrow) was obtained from 6-10 weekold male Balb/C or C57BU6 mice. The marrow was then washed with RPMI+10%FBS (JRH, Lenexa KS; Hyclone, Logan Utah) and suspended in RPMI+10% FBSas a whole marrow cell suspension. The whole marrow cell suspension wasthen subjected to a density gradient (Nycoprep, 1.077, Animal; GibcoBRL) to enrich for low density, mostly mononuclear, cells as follows:The whole marrow cell suspension (About 8 ml) was carefully pipeted ontop of about 5 ml Nycoprep gradient solution in a 15 ml conical tube,and then centrifuged at 600×g for 20 minutes. The interface layer,containing the low density mononuclear cells, was then removed, washedwith excess RPMI+10% FBS, and pelleted by centrifugation at 400×g for5-10 minutes. This pellet was resuspended in RPMI+10% FBS and plated ina T-75 flask at approximately 106 cells/ml, and incubated at 37° C. 5%CO₂ for approximately 2 hours. The resulting cells in suspension wereNon-Adherent Low Density (NA LD) Marrow Cells.

[0280] B. 96-Well Assay

[0281] NA LD Mouse Marrow Cells were plated at 25,000 to 45,000cells/well in 96 well tissue culture plates in RPMI +10% FBS+1 ng/mLmouse Stem Cell Factor (mSCF) (R&D Systems, Minneapolis, Minn.), plus 5%conditioned medium from one of the following: (1) 293 cells expressingadenoviral zsig81, or (2) adenovirus infected 293 cells not expressingzsig81. These cells were then subjected to a variety of cytokinetreatments to test for expansion or differentiation of hematopoieticcells from the marrow. To test, the plated NA LD mouse marrow cells weresubjected to mouse Interleukin 4 (mIL-4), mouse Macrophage-Colonystimulating factor (mM-CSF) (R&D Systems), or one of a panel of othercytokines (R&D Systems). Serial dilution of mIL-4, mM-CSF, or the othercytokines, were tested, with 2-fold serial dilution from about 50 ng/mldown to about 6.25 ng/ml concentration. After 8 to 12 days the 96-wellassays were scored for cell proliferation by Alamar blue assay (Accumed,Chicago, Ill.).

[0282] C. Mouse Bone Marrow Assays Using Human zSig81.CEE Isolated FromBaculovirus.

[0283] NALD mouse bone marrow cells were plated at 200,000 cells/well in24 well tissue culture dishes. The culture medium was supplemented with2 ng/mL stem cell factor (R&D systems, Minn., Minn.), plus one of thefollowing: 1) 15 ng/mL murine IL-4 (R&D systems, Minn., Minn.) 2) 15ng/mL murine GM-CSF 3) 50 ng/mL human zSig81.CEE 4) nothing 5) 15 ng/mLmurine IL-4 (R&D systems, Minn., Minn.) and 50 ng/mL human zSig81.CEE 6)15 ng/mL murine GM-CSF (R&D systems, Minn., Minn.) and 50 ng/mL humanzSig81.CEE. At day 6 following initiation there was significantoutgrowth in cultures containing murine zSig81, IL-4, or GM-CSF alone,or in combination. In addition, cells cultured in the presence zSig81displayed morphologies that were absent in cultures not containingzSig81.

[0284] In another experiment, NALD mouse bone marrow cells were plated200,000 cells/well in 24-well tissue culture dishes. The culture mediumwas supplemented with 2 ng/ml stem cell factor (R&D Systems, Minn.Minn.), plus one of the following: (1) 15 ng/ml murine IL-4 (R&DSystems), (2) 2% conditioned medium (CM) from 293 cells expressingmzSig81, (3) 2% CM from adenovirus infected 293 cells not expressingzsig81, (4) nothing, (5) 15 ng/ml murine IL4 (R&D Systems) plus 2% CMfrom 293 cells expressing murine zsig81, (6) 15 ng/ml mIL-4 (R&DSystems) plus 2% CM from adenovirus infected 293 cells. At culture days4 and 6, the cultures containing zsig81 or IL-4 were much denser thancultures containing SCF or control CM. Furthermore, the cells culturedin the presence of mzsig81 had a different morphology from thosecultured in the absence of mzsig81. The cells from cultures (2), (4),(5) and (6) were removed and stained with antibodies against B220(Pharmingen, San Diego, Calif.) or CD-80 (Pharmingen). There were noB220 positive cells observed in any of the cultures. Cells exposed tomzsig81 were positive for CD-80 and cells not exposed to mzsig81 werenegative for CD-80. These data suggest that cells proliferating in thepresence of zsig81 are of the dendritic lineage.

[0285] Human bone marrow cells were plated at 800,00 cells/well in12-well culture dishes containing culture medium with 2.5 mg/ml FLT-3(R&D Systems) and of the following: (1) nothing, (2) hIL-4 (R&DSystems), (3) BHK CM containing hzsig81, (4) BHK CM, (5) hIL-4 (R&DSystems) and BHK CM. At day 12 of culturing, the cells cultured in thepresence of IL-4, hzsig81, and hzsig81 plus IL-4, all displayed similarmorphologies that were not seen in cultures not exposed to hzsig81 orIL-4. In addition, cultures containing hzsig81 contained more cells. Atday 12, the cells were removed and stained antibodies against CD80(Pharmingen), CD11c (Pharmingen), or HLA-DR (Pharmingen). Cells culturedin the presence of hzsig81 showed increased staining for CD-80, CD11c,and HLA-DR. The increase was equal to that seen for cells cultured inthe presence of hIL-4, and was not seen in control cultures containingBHK CM without zsig81 or IL-4. The data suggest that the proliferatingcells are dendritic lineage because these markers are found on maturedendritic cells.

[0286] Additional evidence that zsig81 targets dendritic cells was seenwhen iodinated zsig81 was found to bind to an immature dendritic cellsline (JAWS II, ATCC No. CRL-1194). The binding was inhibited in a dosedependent manner by unlabeled hzsig81, but not by other cytokines.

Example 11 Mixed Lymphocyte Reaction (MLR)

[0287] A. Preparation of Stimulator Cells (Dendritic Cells)

[0288] A mouse dendritic cell line, JAWS II (CRL-11904; American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209) is grown to high density (1-2×10 ⁶ cells/ml) in α-MEM(Minimal Essential Medium, alpha-modification, containing 10% fetalbovine serum (FBS), 1 mM sodium pyruvate, 4 mM glutamine)+5 ng/ml murineGM-CSF. Additional cytokines used to activate the cells includeinterferon-y (100 U/ml), tumor necrosis factor-α (10 ng/ml), andinterleukin-4 (10 ng/ml). The culture supernatant containing thenonadherent cells is pooled with adherent cells removed by washing withVersene and the cells are resuspended at 3×10⁵ cells/ml in RPMI-1640medium (containing 10% FBS, 10 mM HEPES, 4 mM glutamine, 5.7×10⁻⁵ M2-mercaptoethanol, 50 μg/ml gentamycin, 100 U/ml penicillin, 100 μg/mlstreptomycin).

[0289] Splenic dendritic cells are isolated by the method of Swiggard etal. (Curr. Protocols Immunol. 3.7.1-3.7.11, 1992) from spleens ofC57Bl/6 and BALB/c mice. Briefly, single cell suspensions of spleencells are generated by digestion with collagenase and a low densityfractionation. The low density fraction is obtained by centrifugation ofthe cells through a low density solution (refractive index ofapproximately 1.364) of bovine serum albumin (BSA) in phosphate-bufferedsaline (PBS) onto a high density cushion (refractive index ofapproximately 1.385) of BSA in PBS and contains primarily dendriticcells, macrophages, and some B cells. Cells are resuspended at 37° C. inRPMI medium at 1×10⁷ cells/ml and 4 ml of the suspension is plated per60 mm tissue culture plate. After a 90 minute incubation at 37° C.,nonadherent cells are gently removed, adherent cells are washed withRPMI, and incubated in RPMI for an additional 30-60 min. Nonadherentcells are again removed and adherent cells gently washed with RPMI andincubated in RPMI for 12-20 hours at 37° C . Splenic dendritic cellsdetached during the final incubation and are isolated as nonadherentcells. The nonadherent splenic dendritic cells are resuspended in RPMIat 3×10⁵ cells/ml.

[0290] JAWS II and splenic dendritic stimulator cells are irradiated for40 minutes in a ¹³⁷Cs irradiator (Gammacell 40, Nordion InternationalInc., Kanata, Ontario, Canada) at 550 rads/min before use in the MLR.

[0291] B. Preparation of Responder Cells (T Cells)

[0292] Spleens and lymph nodes are removed from C57Bl/6 or BALB/c mice(Jackson Labs, Bar Harbor, Me.). Spleen cell suspensions in BSS-BSAbuffer are made by mechanical disruption of the spleen between glassslides. Red blood cells are lysed by resuspending the spleen cell pelletin 0.9 ml dH₂O followed quickly by addition of 0.1 ml 10×HBSS. Lymphnode cell suspensions in BSS are made by teasing the nodes with sterileforceps and are pooled with the autologous spleen cell suspension andfiltered through nylon cloth filters to remove debris.

[0293] The single cell suspension of spleen and lymph node cells isloaded onto a nylon wool column pre-equilibrated at 37° C. with BSS+5%FBS. After incubation at 37° C. for 45 minutes, the T cells are elutedwith 37° C. BSS+5% FBS (12 ml per 1.5 g nylon wool column loaded withapproximately 1.5×10⁸ total spleen+lymph node cells). The T cells(usually 80-90% pure) are resuspended in RPMI at 3×10⁶ cells/ml.

[0294] C. Incubation Conditions for MLR

[0295] 3×10⁵ responder cells per well (96-well plate) are mixed induplicate with increasing numbers of irradiated stimulator cells(usually 3×10³, 1×10⁴, 3×10⁴ cells) in a final volume of 200 μl.Controls includes responder cells alone and stimulator cells alone. Asyngeneic MLR includes responder and stimulator cells from the samemouse strain (e.g., C57Bl/6 or BALB/c), whereas an allogeneic MLR hasstimulator cells incubated with responder cells from a different strain(e.g., C57Bl/6 or JAWS II stimulator cells with BALB/c responder cells).The MLR cultures are incubated at 37° C. for approximately 72-76 hoursbefore addition of 1 μCi/well ³H-thymidine to assay proliferation ofresponder cells. Cultures are harvested 16-20 hours later with a Skatroncell harvester (Skatron, Sterling, Va.), and the incorporated³H-thymidine is determined with a Wallac Betaplate liquid scintillationcounter (Pharmacia).

[0296] When JAWS II cells are induced with a combination of factors thatstimulate allogeneic T cells, the JAWS II cells proliferate. JAWS IIcells are induced with TNF-α, IFN-γ, GM-CSF and IL-4. In addition, theJAWS II cell line do not stimulate proliferation in syngeneic T cells.

[0297] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

1 36 1 1600 DNA Homo sapiens CDS (134)...(655) sig_peptide (134)...(184)1 gaattcggct cgaggcaaaa ggaagggagg gaagcactcc atcatctcac tgggaagaac 60ggcacgggca tacctgcagc tactggggtt ccactgggct tgagggtcga tttttcacct 120tttgaaggac aag atg cat tgg aag atg ttg ctg ctt ctg ctg ttg tat 169 MetHis Trp Lys Met Leu Leu Leu Leu Leu Leu Tyr -15 -10 tac aat gct gag gcttct atg tgc cac agg tgg agc agg gct gtg ctc 217 Tyr Asn Ala Glu Ala SerMet Cys His Arg Trp Ser Arg Ala Val Leu -5 1 5 10 ttc cct gcc gcc caccgg cca aag agg tcc tca tca ctg cca ttg aac 265 Phe Pro Ala Ala His ArgPro Lys Arg Ser Ser Ser Leu Pro Leu Asn 15 20 25 cca gtc ctg cag acc tccctg gag gag gtg gag ctg ctc tac gag ttc 313 Pro Val Leu Gln Thr Ser LeuGlu Glu Val Glu Leu Leu Tyr Glu Phe 30 35 40 ctg ctg gcc gaa ctt gag atcagc cct gac ctg cag atc tcc atc aag 361 Leu Leu Ala Glu Leu Glu Ile SerPro Asp Leu Gln Ile Ser Ile Lys 45 50 55 gac gag gag ctg gcc tcc ttg cggaag gcc tca gac ttc cgc acc gtc 409 Asp Glu Glu Leu Ala Ser Leu Arg LysAla Ser Asp Phe Arg Thr Val 60 65 70 75 tgc aac aac gtc atc ccc aag agcatc cca gac atc cgc cgg ctc agc 457 Cys Asn Asn Val Ile Pro Lys Ser IlePro Asp Ile Arg Arg Leu Ser 80 85 90 gcc agc ctc tcc agc cac cct ggc atcctc aag aaa gaa gac ttt gaa 505 Ala Ser Leu Ser Ser His Pro Gly Ile LeuLys Lys Glu Asp Phe Glu 95 100 105 agg aca gtg ctg acc ctg gcc tac acagcc tac cgc aca gcc ctg tcc 553 Arg Thr Val Leu Thr Leu Ala Tyr Thr AlaTyr Arg Thr Ala Leu Ser 110 115 120 cac ggc cat cag aag gac atc tgg gcgcag tcc ctc gtt agc ctc ttc 601 His Gly His Gln Lys Asp Ile Trp Ala GlnSer Leu Val Ser Leu Phe 125 130 135 cag gcc ctg agg cac gac ttg atg cgctcc tca cag ccg gga gta cct 649 Gln Ala Leu Arg His Asp Leu Met Arg SerSer Gln Pro Gly Val Pro 140 145 150 155 ccc tga gagactggcc cacaccaggacctcagagca gggaccagca cagtaatcca 705 Pro gaaagtcttc attctctactccatttacag agaccagcaa caaaacactt accgctgaca 765 cagagcagca gagatcaaacagtaaccccg atgctctttt ctccttgtag tttcctggaa 825 gacacatctg attcatgccatcatgtgacc tgggctggaa gaaagggctg gaatggtcat 885 tcaagacgcc tccatgggcagaatggtttg cctatggcag gcagaattct gatatgcttc 945 aacccagagc agtggccacacactcaagag tgagaacagg cgtgagccac cgtgcctggc 1005 ccaggatcta aaaactttctaagtttcctc catcgttggc atcctcacag ctatctccaa 1065 tgtcactcaa gagacatcaacagacattta actgctgcag acttcattgc tctgtcacct 1125 caccttgaat ctaacaaatcaaagtatttc tgcaggtcca atggtctaaa atcaaatgct 1185 tgttaaatga ctttttacaacaccccttac tttcctaatc catttcaatc ttattttttt 1245 tattgtggta aaaaacacatcacgtaaaat gtaccatctt aaccattttt aagcatatgg 1305 tacagcagtg ttaactccatgcatgttgtg aaacagaccc ccggaacttt ctcatcttgt 1365 aattctgaag ttctatacccaccgaacaac tcctcttttc cccttccccc tgcctgcccc 1425 agctcttggc accattattctgctttctgt ttttgagagt ctgactactt aagatacctc 1485 atacaagcgg gatctggcttacatttcttg agcattgtat tctggaaaag tgtttccttc 1545 ctctgaaaaa tgggtagagttctgaaggag aactactggt cttattgtac acttg 1600 2 173 PRT Homo sapiensSIGNAL (1)...(17) 2 Met His Trp Lys Met Leu Leu Leu Leu Leu Leu Tyr TyrAsn Ala Glu -15 -10 -5 Ala Ser Met Cys His Arg Trp Ser Arg Ala Val LeuPhe Pro Ala Ala 1 5 10 15 His Arg Pro Lys Arg Ser Ser Ser Leu Pro LeuAsn Pro Val Leu Gln 20 25 30 Thr Ser Leu Glu Glu Val Glu Leu Leu Tyr GluPhe Leu Leu Ala Glu 35 40 45 Leu Glu Ile Ser Pro Asp Leu Gln Ile Ser IleLys Asp Glu Glu Leu 50 55 60 Ala Ser Leu Arg Lys Ala Ser Asp Phe Arg ThrVal Cys Asn Asn Val 65 70 75 Ile Pro Lys Ser Ile Pro Asp Ile Arg Arg LeuSer Ala Ser Leu Ser 80 85 90 95 Ser His Pro Gly Ile Leu Lys Lys Glu AspPhe Glu Arg Thr Val Leu 100 105 110 Thr Leu Ala Tyr Thr Ala Tyr Arg ThrAla Leu Ser His Gly His Gln 115 120 125 Lys Asp Ile Trp Ala Gln Ser LeuVal Ser Leu Phe Gln Ala Leu Arg 130 135 140 His Asp Leu Met Arg Ser SerGln Pro Gly Val Pro Pro 145 150 155 3 1547 DNA Mus musculus CDS(212)...(733) sig_peptide (212)...(262) 3 ggattcggca cgagggagaggtaccaactt ctgtcccacc caagaggctg catccgcctc 60 catcctgtgg agccagggagaggcccttgc tttccttata gacaagaaag ggcagtaaga 120 actctgtcct ctcgctgagaagagcagggg tccacctgca gcccctgggg tcccgcagga 180 atagaaggtc agcttgtctccctcctggaa g atg tcc tgg aag gcg ctg acg 232 Met Ser Trp Lys Ala Leu Thr-15 att ctg ctg gta ttc tcc agc acc cag gcc act gcg tcc tgc agg tgg 280Ile Leu Leu Val Phe Ser Ser Thr Gln Ala Thr Ala Ser Cys Arg Trp -10 -5 15 agc agg gcc gca ctg ttc cca gct gcc cat cgg cca aag agg tcc ttg 328Ser Arg Ala Ala Leu Phe Pro Ala Ala His Arg Pro Lys Arg Ser Leu 10 15 20tca ctg cca ttg aat cca gtc ctg cag acc tcc ctg gag gag gtg gaa 376 SerLeu Pro Leu Asn Pro Val Leu Gln Thr Ser Leu Glu Glu Val Glu 25 30 35 ctgctg tat gag ctc ttg cta gct gaa att gag atc agc cca gac ctg 424 Leu LeuTyr Glu Leu Leu Leu Ala Glu Ile Glu Ile Ser Pro Asp Leu 40 45 50 gag atctcc atc aag gac gag gag cta gct tcc ctg cgg aag gcc ttg 472 Glu Ile SerIle Lys Asp Glu Glu Leu Ala Ser Leu Arg Lys Ala Leu 55 60 65 70 agt ttccac tca atc tgc aat aac ata atc ccc aag cgt atc cca gat 520 Ser Phe HisSer Ile Cys Asn Asn Ile Ile Pro Lys Arg Ile Pro Asp 75 80 85 atc cga aggctg agt gcc aac ctg gca aac cac cct gga atc ctc aag 568 Ile Arg Arg LeuSer Ala Asn Leu Ala Asn His Pro Gly Ile Leu Lys 90 95 100 aaa gaa gacttt gag agg ata aca tta acc ctg gcg tac aca gcc tat 616 Lys Glu Asp PheGlu Arg Ile Thr Leu Thr Leu Ala Tyr Thr Ala Tyr 105 110 115 cgg aca gcctta tct gaa ggg cat cag aag gac atc tgg gct cag tcc 664 Arg Thr Ala LeuSer Glu Gly His Gln Lys Asp Ile Trp Ala Gln Ser 120 125 130 ctc atc agccta ttc cag gcc ctg agg cat gac ttg atg cgg tcc tcg 712 Leu Ile Ser LeuPhe Gln Ala Leu Arg His Asp Leu Met Arg Ser Ser 135 140 145 150 agc cctgct gtg tca tcc tga gagaatggct catgctagaa ctttgaagca 763 Ser Pro Ala ValSer Ser 155 ggaacaggca cacacagtct tctagaactt tcatcctcta ctgcactttcagagaaaagt 823 atatacttcc cacacagaat agcaaagata aatgagtcac cccaatattttttgtccctt 883 gttgcttcca gacagacata tccgacctat gttataatgt tacctgagaaaaggctagac 943 tggactttca agatgcctcc agaggccaac tggtctacct ggtaatgagcagacttctga 1003 gatatactta cacacatacc caagagtagg gactgaggat ggagtctgagcatggcagga 1063 ggatggtggg cagattcctt tggttctaag ggatctgtgt tgaatgaatattttctggca 1123 ggttctatgg taaatataaa aaaggcagag atgcattcaa attaatatgctattagccaa 1183 gaaggatata cttggcttgc cccaaagcca tgaagaagac tctgtattttggtgacctac 1243 ttgacttggt ggaaatgcta gcagtccacc catgccctat catttcaatgtagaagccag 1303 gctaaagcat agtgccttcc taatgaaaga ggtaacacca ctatgcgtgtttttcctaaa 1363 ataccatagc actgtcagcg acttgggtgc tcctaaaaaa attcgctttcagatgacaga 1423 ttgtttacct ttcaaatgct gatttttttt ctttcaaaat gttagtttgatatctgttca 1483 ttatttatat taaatactgg ttgatattta aaaaaaaaaa aaaaaaaaaaaccattgcgg 1543 ccgc 1547 4 173 PRT Mus musculus SIGNAL (1)...(17) 4 MetSer Trp Lys Ala Leu Thr Ile Leu Leu Val Phe Ser Ser Thr Gln -15 -10 -5Ala Thr Ala Ser Cys Arg Trp Ser Arg Ala Ala Leu Phe Pro Ala Ala 1 5 1015 His Arg Pro Lys Arg Ser Leu Ser Leu Pro Leu Asn Pro Val Leu Gln 20 2530 Thr Ser Leu Glu Glu Val Glu Leu Leu Tyr Glu Leu Leu Leu Ala Glu 35 4045 Ile Glu Ile Ser Pro Asp Leu Glu Ile Ser Ile Lys Asp Glu Glu Leu 50 5560 Ala Ser Leu Arg Lys Ala Leu Ser Phe His Ser Ile Cys Asn Asn Ile 65 7075 Ile Pro Lys Arg Ile Pro Asp Ile Arg Arg Leu Ser Ala Asn Leu Ala 80 8590 95 Asn His Pro Gly Ile Leu Lys Lys Glu Asp Phe Glu Arg Ile Thr Leu100 105 110 Thr Leu Ala Tyr Thr Ala Tyr Arg Thr Ala Leu Ser Glu Gly HisGln 115 120 125 Lys Asp Ile Trp Ala Gln Ser Leu Ile Ser Leu Phe Gln AlaLeu Arg 130 135 140 His Asp Leu Met Arg Ser Ser Ser Pro Ala Val Ser Ser145 150 155 5 519 DNA Artificial Sequence Degenerate sequence derivedfrom human zsig81 protein sequence 5 atgcaytgga aratgytnyt nytnytnytnytntaytaya aygcngargc nwsnatgtgy 60 caymgntggw snmgngcngt nytnttyccngcngcncaym gnccnaarmg nwsnwsnwsn 120 ytnccnytna ayccngtnyt ncaracnwsnytngargarg tngarytnyt ntaygartty 180 ytnytngcng arytngarat hwsnccngayytncarathw snathaarga ygargarytn 240 gcnwsnytnm gnaargcnws ngayttymgnacngtntgya ayaaygtnat hccnaarwsn 300 athccngaya thmgnmgnyt nwsngcnwsnytnwsnwsnc ayccnggnat hytnaaraar 360 gargayttyg armgnacngt nytnacnytngcntayacng cntaymgnac ngcnytnwsn 420 cayggncayc araargayat htgggcncarwsnytngtnw snytnttyca rgcnytnmgn 480 caygayytna tgmgnwsnws ncarccnggngtnccnccn 519 6 15 PRT Artificial Sequence variant derived from humanhelix A 6 Xaa Gln Thr Xaa Xaa Glu Glu Xaa Glu Leu Xaa Xaa Glu Phe Xaa 15 10 15 7 15 PRT Artificial Sequence variant derived from human helix B7 Xaa Ser Ile Xaa Xaa Glu Glu Xaa Ala Ser Xaa Xaa Lys Ala Xaa 1 5 10 158 9 PRT Artificial Sequence variant derived from human helix C 8 Asp XaaArg Arg Xaa Xaa Ala Ser Xaa 1 5 9 15 PRT Artificial Sequence variantderived from human helix D 9 Xaa Val Ser Xaa Xaa Gln Ala Xaa Arg His XaaXaa Met Arg Xaa 1 5 10 15 10 22 DNA Artificial Sequence oligonucleotideprimer ZC21621 10 gggctgtgcg gtaggctgtg ta 22 11 22 DNA ArtificialSequence oligonucleotide primer ZC21622 11 aagaacggca cgggcatacc tg 2212 18 DNA Artificial Sequence oligonucleotide primer ZC22801 12aacggcacgg gcatacct 18 13 18 DNA Artificial Sequence oligonucleotideprimer ZC22802 13 gaagcctcag cattgtaa 18 14 25 DNA Artificial Sequenceoligonucleotide primer ZC17251 14 tctggacgtc ctcctgctgg tatag 25 15 25DNA Artificial Sequence oligonucleotide primer ZC17252 15 ggtatggagcaaggggcaag ttggg 25 16 27 DNA Artificial Sequence oligonucleotide primerZC17156 16 gagtggcaac ttccagggcc aggagag 27 17 27 DNA ArtificialSequence oligonucleotide primer ZC17157 17 cttttgctag cctcaaccct gactatc27 18 18 DNA Artificial Sequence oligonucleotide primer ZC21229 18ggccagcaga acgaagca 18 19 18 DNA Artificial Sequence oligonucleotideprimer ZC21711 19 cctgcctgcc gtttcttc 18 20 8 PRT Artificial SequenceFLAG peptide tag 20 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 21 31 DNAArtificial Sequence oligonucleotide primer ZC26461 21 tcagtcgtccggaaggtgga gcagggctgt g 31 22 52 DNA Artificial Sequence oligonucleotideprimer ZC26475 22 cgactgatct agactagtcc atcggcatgt attcgggagg tactcccggctg 52 23 17 DNA Artificial Sequence oligonucleotide primer ZC2359 23agggacctga gcgagtc 17 24 33 DNA Artificial Sequence oligonucleotideprimer ZC12581 24 tcgagctact tatcgtcatc gtccttatag tcg 33 25 27 DNAArtificial Sequence oligonucleotide primer ZC23433 25 ctccggatccatgtcctgga aggcgct 27 26 31 DNA Artificial Sequence oligonucleotideprimer ZC23432 26 gcatgagcca tctagacagg atgacacagc a 31 27 17 DNAArtificial Sequence oligonucleotide primer ZC447 27 taacaatttc acacagg17 28 18 DNA Artificial Sequence oligonucleotide primer ZC976 28cgttgtaaaa cgacggcc 18 29 1635 DNA Homo sapiens 29 atgaaaatcg aagaaggtaaactggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaaattcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaaattcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccgctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttccaggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgcttacccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgccaaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgcgctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacgggggttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgctggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgcagacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccatcaacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggtactgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtattaacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgactgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtcttacgaggaa gagttggcga aagatccacg tattgccgcc 960 accatggaaa acgcccagaaaggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgcggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagactaattcgagc tcccaccatc accatcacca cgcgaattcg 1140 gtaccgctgg ttccgcgtggatcctctatg tgccacaggt ggagcagggc tgtgctcttc 1200 cctgccgccc accggccaaagaggtcctca tcactgccat tgaacccagt cctgcagacc 1260 tccctggagg aggtggagctgctctacgag ttcctgctgg ccgaacttga gatcagccct 1320 gacctgcaga tctccatcaaggacgaggag ctggcctcct tgcggaaggc ctcagacttc 1380 cgcaccgtct gcaacaacgtcatccccaag agcatcccag acatccgccg gctcagcgcc 1440 agcctctcca gccaccctggcatcctcaag aaagaagact ttgaaaggac agtgctgacc 1500 ctggcctaca cagcctaccgcacagccctg tcccacggcc atcagaagga catctgggcg 1560 cagtccctcg ttagcctcttccaggccctg aggcacgact tgatgcgctc ctcacagccg 1620 ggagtacctc cctga 163530 64 DNA Artificial Sequence oligonucleotide primer ZC22990 30tcaccacgcg aattcggtac cgctggttcc gcgtggatcc tctatgtgcc acaggtggag 60cagg 64 31 61 DNA Artificial Sequence oligonucleotide primer ZC22991 31tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca tcagggaggt actcccggct 60 g61 32 40 DNA Artificial Sequence oligonucleotide primer ZC19372 32tgtcgatgaa gccctgaaag acgcgcagac taattcgagc 40 33 60 DNA ArtificialSequence oligonucleotide primer ZC19351 33 acgcgcagac taattcgagctcccaccatc accatcacca cgcgaattcg gtaccgctgg 60 34 60 DNA ArtificialSequence oligonucleotide primer ZC19352 34 actcactata gggcgaattgcccgggggat ccacgcggaa ccagcggtac cgaattcgcg 60 35 42 DNA ArtificialSequence oligonucleotide primer ZC19371 35 acggccagtg aattgtaatacgactcacta tagggcgaat tg 42 36 5 PRT Artificial Sequence Glu Glu peptidetag 36 Glu Tyr Pro Met Glu 1 5

We claim:
 1. An isolated polypeptide comprising at least nine contiguousamino acid residues of SEQ ID NO:
 2. 2. The isolated polypeptide ofclaim I comprising at least 15 amino acid residues.
 3. An isolatedpolypeptide comprising a sequence of amino acid residues selected fromthe group consisting of: (a) residues 30-44 of SEQ ID NO: 2; (b)residues 56-70 of SEQ ID NO: 2; (c) residues 86-94 of SEQ ID NO: 2; and(d) residues 135-149 of SEQ ID NO:
 2. 4. The isolated polypeptide ofclaim 3, wherein (c) comprises residues 80-94 or 86-100 of SEQ ID NO: 2.5. An isolated polypeptide comprising a sequence of amino acid residuesthat is at least 90% identical to amino acid residues 30 to 149 of SEQID NO:
 2. 6. The polypeptide of claim 5, wherein the sequence of aminoacid residues comprises residues 5 to 156 of SEQ ID NO:
 2. 7. Thepolypeptide of claim 5, wherein the sequence of amino acid residuescomprises residues 1 to 156 of SEQ ID NO:
 2. 8. The polypeptide of claim5, wherein the sequence of amino acid residues comprises residues −17 to156 of SEQ ID NO:
 2. 9. A fusion protein comprising at least twopolypeptides, wherein a first polypeptide is selected from the groupconsisting of: (a) residues 30-44 of SEQ ID NO: 2; (b) residues 56-70 ofSEQ ID NO: 2; (c) residues 86-94 of SEQ ID NO: 2; and (d) residues135-149 of SEQ I) NO:
 2. 10. The fusion protein of claim 9, wherein asecond polypeptide is selected from a functional fragment of anothercytokine or a toxin conjugate.
 11. A fusion protein comprising a firstpolypeptide and a second polypeptide, joined by a peptide bond, saidfirst polypeptide comprises a signal sequence and a second polypeptidecomprising an a sequence of amino acids as shown in SEQ ID NO: 2 fromamino acid residues 30-149.
 12. A fusion protein comprising a firstpolypeptide and a second polypeptide, joined by a peptide bond, whereinthe first polypeptide is a maltose binding protein, the peptide bond isselected from the group consisting of Factor Xa cleavage site, thrombincleavage site or enterokinase cleavage site, and the second polypeptidecomprising an a sequence of amino acids as shown in SEQ ID NO: 2 fromamino acid residues 30-149.
 13. A composition comprising a sequence ofamino acid residues selected from the group consisting of: (a) residues30-44 of SEQ ID NO: 2; (b) residues 56-70 of SEQ ID NO: 2; (c) residues86-94 of SEQ ID NO: 2; and (d) residues 135-149 of SEQ ID NO: 2; and apharmaceutically acceptable vehicle.
 14. An isolated polynucleotideencoding a polypeptide comprising: (a) residues 30-44 of SEQ ID NO: 2;(b) residues 56-70 of SEQ ID NO: 2; (c) residues 86-94 of SEQ ID NO: 2;or (d) residues 135-149 of SEQ ID NO:
 2. 15. The isolated polynucleotideof claim 14 comprising: (a) nucleotides 272-316 of SEQ ID NO: 1; (b)nucleotides 350-394 of SEQ ID NO: 1; (c) nucleotides 440-466 of SEQ IDNO: 1; or (d) nucleotides 587-631 of SEQ ID NO:
 1. 16. The isolatedpolynucleotide of claim 14 comprising: (a) nucleotides 139-183 of SEQ IDNO: 5; (b) nucleotides 216-261 of SEQ ID NO: 5; (c) nucleotides 307-331of SEQ ID NO: 5; or (d) nucleotides 454-499 of SEQ ID NO:
 5. 17. Anisolated polynucleotide encoding a polypeptide comprising a sequence ofamino acid residues that is at least 90% identical to amino acidresidues 30 to 149 of SEQ ID NO: 2
 18. An isolated polynucleotidecomprising a sequence of nucleotides as shown in SEQ ID NO: 1 fromnucleotide 272 to nucleotide
 631. 19. The polynucleotide of claim 18,wherein the sequence of nucleotides comprises the sequence as shown inSEQ ID NO: 1 from nucleotide 185 to nucleotide
 655. 20. Thepolynucleotide of claim 18, wherein the sequence of nucleotidescomprises the sequence as shown in SEQ ID NO: 1 from nucleotide 134 tonucleotide
 655. 21. An expression vector comprising the followingoperably linked elements: a transcription promoter; a DNA segmentencoding the polynucleotide of claim 14; and a transcription terminator.22. A cultured cell into which has been introduced the expression vectorof claim 21, wherein said cell expresses said DNA segment.
 23. A methodof making a polypeptide comprising: culturing the cell of claim 22 underconditions whereby the DNA segment is expressed and the polypeptide isproduced; and recovering the polypeptide.
 24. An antibody whichspecifically binds to a polypeptide of claim
 1. 25. A method forexpansion of hematopoietic cells and hematopoietic cell progenitorscomprising culturing bone marrow or peripheral blood cells with acomposition comprising an amount of zsig81 polypeptide sufficient toproduce an increase in the number of hematopoietic cells in the bonemarrow or peripheral blood cells as compared to bone marrow orperipheral blood cells cultured in the absence of zsig81.
 26. The methodof claim 25, wherein the hematopoietic cells and hematopoietic cellprogenitors are lymphoid cells.
 27. The method of claim 25, wherein thehematopoietic cells and hematopoietic progenitor cells are dendriticcells.
 28. The method of modulating an immune response in a mammalexposed to an antigen comprising: (1) determining a level ofantigen-specific antibody; (2) administering a composition comprisingzsig81 polypeptide in a pharmaceutically acceptable vehicle; (3)determining a post administration level of antigen-specific antibody;(4) comparing the level of antibody in step (1) to the level of antibodyin step (3), wherein a change in antibody level is indicative ofmodulating the immune response.
 29. The method of claim 25, wherein thehematopoietic cells and hematopoietic progenitor cells are myeloidcells.
 30. A method of detecting the presence of zsig81 RNA in abiological sample, comprising the steps of: (a) contacting a zsig81nucleic acid probe under hybridizing conditions with either (i) test RNAmolecules isolated from the biological sample, or (ii) nucleic acidmolecules synthesized from the isolated RNA molecules, wherein the probehas a nucleotide sequence of nucleic acid molecule of claim 20, or itscomplement; and (b) detecting the formation of hybrids of the nucleicacid probe and either the test RNA molecules or the synthesized nucleicacid molecules, wherein the presence of the hybrids indicates thepresence of zsig81 in the biological sample.
 31. A method of detectingthe presence of zsig81 in a biological sample, comprising the steps of:(a) contacting the biological sample with an antibody, or an antibodyfragment, of claim 24, wherein the contacting is performed underconditions that allow the binding of the antibody or antibody fragmentto the biological sample; and (b) detecting any of the bound antibody orbound antibody fragment.
 32. A method for stimulating antigenic responseto tumor antigens comprising the steps of: (1) isolating hematopoieticcells from a mammal; (2) exposing the isolated hematopoietic cells to atumor antigen; (3) culturing the exposed cells in a compositioncomprising an isolated polypeptide of at least nine contiguous aminoacid residues of SEQ ID NO: 2; and (4) administering the cultured cellsback to the mammal.